Anti-OX40 binding proteins

ABSTRACT

The present disclosure provides anti-OX40 antibodies, and antigen-binding portions thereof. In certain embodiments, the antibodies or fragments thereof, are used for the treatment of cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Stage Application of InternationalApplication No. PCT/US2017/045788, filed Aug. 7, 2017, which claimspriority to U.S. Provisional Application No. 62/371,993, filed on Aug.8, 2016, and entitled “Anti-OX40 Binding Proteins”, the entire contentsof each of which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web and is herebyincorporated by reference in its entirety. Said ASCII copy, created onFeb. 7, 2019, is named Sequence_Listing_S103014_2040US.PCT.txt and is100 kilobytes in size.

BACKGROUND

OX40 (also known as CD 134, TNFRSF4, ACT35 or TXGP1L) is a member of theTNF receptor superfamily, which includes 4-1BB, CD27, CD30 and CD40. Theextracellular ligand binding domain of OX40 is composed of 3 fullcysteine-rich domains (CRDs) and a partial, fourth C-terminal CRD(Bodmer et al, 2002, Trends Biochem. Sci., 27, 19-26). The ligand forOX40, OX40L, is a member of the TNF family and is expressed on activatedantigen presenting cells (APC), including B cells, macrophages,endothelial cells and dendritic cells (DC). OX40 is a membrane-boundreceptor; however a soluble isoform has also been detected (Taylor andSchwarz, 2001, J. Immunol. Methods, 255, 67-72). OX40 is not expressedon resting T cells, but is transiently expressed on activated T cellsafter ligation of the T cell receptor (TCR).

OX40 is a major costimulatory receptor with sequential engagement ofCD28 and OX40 resulting in optimal T cell proliferation and survival.Ligation of OX40 on activated T cells leads to enhanced cytokineproduction and proliferation of both CD4+ and CD8+ T cells (Gramaglia etal., 2000, J. Immunol, 165, 3043-3050, Bansal-Pakala et al., 2004, J.Immunol., 172, 4821-425) and can contribute to both ongoing Th1 and Th2responses (Gramaglia et al., 1998, J. Immunol., 161, 6510-6517,Arestides et al, 2002, Eur. J. Immunol. 32, 2874-2880). OX40costimulation prolongs T cell survival beyond the initial effector phaseof the immune response and increases the number of memory T cellsthrough inhibition of effector T cell death.

When immune activation is excessive or uncontrolled, pathologicalallergy, asthma, inflammation, autoimmune and other related diseases mayoccur.

Tumor cells commonly ‘escape’ the immune system by induction of anactive immune tolerance largely mediated by regulatory T lymphocytes(Tregs et al. Immunol Rev. 2011; 241:104-118). Therefore, the balancebetween effector (i.e., direct or indirect eradication of tumor cells) Tlymphocytes (Teffs) and tolerogenic (i.e., suppression of Teffs effectorfunction and survival) Tregs appears to be important for effectiveanti-tumor immunotherapy. In other words, an effective anti-tumor immuneresponse can be obtained by enhancing effector function oftumor-specific Teffs and/or by attenuating suppressive function oftumor-specific Tregs. A key receptor that has been shown to mediatethese responses is OX40 (CD134). (Sugamura et al., Nature Rev. Imm.2004; 4: 420-431).

In vivo ligation of mouse CD134 receptor (by either soluble mouse OX40ligand (OX40L)-immunoglobulin fusion proteins or mouse OX40L mimetics,such as anti-mouse CD134-specific antibodies) in tumor-bearing miceenhances anti-tumor immunity, leads to tumor-free survival in mousemodels of various murine malignant tumor cell lines, e.g., lymphoma,melanoma, sarcoma, colon cancer, breast cancer, and glioma (Sugamura etal. Nature Rev. Imm. 2004; 4:420631). Al-Shamkhani et al. (Eur. J. Chem.1996; 26: 1695-1699) used an anti-OX40 antibody called OX86, which didnot block OX40L-binding, in order to explore differential expression ofOX40 on activated mouse T-cells; and Hirschhorn-Cymerman et al. (J. Exp.Med. 2009; 206: 1103-1116) used OX86 together with cyclophosphamide in amouse model as a potential chemoimmunotherapy.

Thus, there remains a need in the art for effective treatments based onOX40, particularly anti-OX40 antibodies.

SUMMARY OF THE INVENTION

The invention provides antibodies that specifically bind to OX40,including human OX40.

In a first aspect, the invention features an isolated anti-OX40antibody, or an antigen-binding fragment thereof, comprising a heavychain variable domain comprising a heavy chain CDR set (CDR1, CDR2, andCDR3) selected from the group consisting of SEQ ID Nos: 49, 50 and 51;SEQ ID Nos: 55, 56 and 57; SEQ ID Nos: 61, 62 and 63; SEQ ID Nos: 67, 68and 69; SEQ ID Nos: 73, 74 and 75; SEQ ID Nos: 79, 80 and 81; SEQ IDNos: 85, 86 and 87; SEQ ID Nos: 91, 92 and 93; SEQ ID Nos: 103, 104 and105; SEQ ID Nos: 109, 110 and 111; SEQ ID Nos: 118, 119 and 120; SEQ IDNos: 133, 134 and 135; SEQ ID Nos: 139, 140 and 141; SEQ ID Nos: 148,149, and 150; SEQ ID Nos: 157, 158 and 159; SEQ ID Nos: 163, 164 and165; SEQ ID Nos: 172, 173 and 174; SEQ ID Nos: 178, 179 and 180; SEQ IDNos: 184, 185 and 186; SEQ ID Nos. 195, 196 and 197; and SEQ ID Nos.203, 204 and 205; and a light chain variable domain comprising a lightchain CDR set (CDR1, CDR2, and CDR3) selected from the group consistingof SEQ ID Nos: 52, 53 and 54; SEQ ID Nos: 58, 59 and 60; SEQ ID Nos: 64,65 and 66; SEQ ID Nos: 70, 71 and 72; SEQ ID Nos: 76, 77 and 78; SEQ IDNos: 82, 83 and 84; SEQ ID Nos: 88, 89 and 90; SEQ ID Nos: 94, 95 and96; SEQ ID Nos: 97, 98 and 99; SEQ ID Nos: 100, 101 and 102; SEQ ID Nos:106, 107 and 108; SEQ ID Nos: 112, 113 and 114; SEQ ID Nos: 115, 116 and117; SEQ ID Nos: 121, 122 and 123; SEQ ID Nos: 124, 125 and 126; SEQ IDNos: 127, 128 and 129; SEQ ID Nos: 130, 131 and 132; SEQ ID Nos: 136,137 and 138; SEQ ID Nos: 142, 143 and 144; SEQ ID Nos: 145, 146 and 147;SEQ ID Nos: 151, 152 and 153; SEQ ID Nos: 154, 155 and 156; SEQ ID Nos:160, 161 and 162; SEQ ID Nos: 166, 167 and 168; SEQ ID Nos: 169, 170 and171; SEQ ID Nos: 175, 176 and 177; SEQ ID Nos: 181, 182 and 183; SEQ IDNos: 187, 188 and 189; SEQ ID Nos: 190, 191 and 192; SEQ ID Nos. 198,199 and 200; SEQ ID Nos. 206, 207 and 208; SEQ ID Nos. 210, 211 and 212;and SEQ ID Nos. 214, 215 and 216. In one embodiment, the heavy chainvariable domain comprises an amino acid sequence that is at least 95%identical to an amino acid sequence selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO.21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ IDNO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQID NO. 193 and SEQ ID NO. 201; and a light chain variable domaincomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213. In oneembodiment, the heavy chain variable domain comprises an amino acidsequence that is at least 96% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42,SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201; and thelight chain variable domain comprises an amino acid sequence that is atleast 96% identical to an amino acid sequence selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ IDNO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38,SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO.47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 andSEQ ID NO. 213. In one embodiment, the heavy chain variable domaincomprises an amino acid sequence that is at least 97% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 1,SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11,SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO.24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ IDNO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 andSEQ ID NO. 201; and the light chain variable domain comprises an aminoacid sequence that is at least 97% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ IDNO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202,SEQ ID NO. 209 and SEQ ID NO. 213. In one embodiment, the heavy chainvariable domain comprises an amino acid sequence that is at least 98%identical to an amino acid sequence selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO.21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ IDNO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQID NO. 193 and SEQ ID NO. 201; and comprises a light chain variabledomain comprising an amino acid sequence that is at least 98% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26,SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213. In oneembodiment, the heavy chain variable domain comprises an amino acidsequence that is at least 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42,SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201; andcomprises a light chain variable domain comprising an amino acidsequence that is at least 99% identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ IDNO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202,SEQ ID NO. 209 and SEQ ID NO. 213. In one embodiment, the heavy chainvariable domain comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201; and comprises a lightchain variable domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ IDNO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23,SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO.30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209and SEQ ID NO. 213.

In a further embodiment, the invention provides an isolated anti-OX40antibody, or an antigen-binding fragment thereof, comprising a heavychain CDR set/light chain CDR set selected from the group consisting ofSEQ ID Nos: 49, 50 and 51/SEQ ID Nos: 52, 53 and 54; SEQ ID Nos: 55, 56and 57/SEQ ID Nos: 58, 59 and 60; SEQ ID Nos: 61, 62 and 63/SEQ ID Nos:64, 65 and 66; SEQ ID Nos: 67, 68 and 69/SEQ ID Nos: 70, 71 and 72; SEQID Nos: 73, 74 and 75/SEQ ID Nos: 76, 77 and 78; SEQ ID Nos: 79, 80 and81/SEQ ID Nos: 82, 83 and 84; SEQ ID Nos: 85, 86 and 87/SEQ ID Nos: 88,89 and 90; SEQ ID Nos: 91, 92 and 93/SEQ ID Nos: SEQ ID Nos: 94, 95 and96; SEQ ID Nos: 67, 68 and 69/SEQ ID Nos: 97, 98, and 99; SEQ ID Nos:55, 56 and 57/SEQ ID Nos: 100, 101 and 102; SEQ ID Nos: 103, 104 and105/SEQ ID Nos: 106, 107 and 108; SEQ ID Nos: 109, 110 and 111/SEQ IDNos: 112, 113 and 114; SEQ ID Nos: 67, 68 and 69/SEQ ID Nos: 115, 116and 117; SEQ ID Nos: 118, 119 and 120/SEQ ID Nos: 121, 122 and 123; SEQID Nos: 67, 68 and 69/SEQ ID Nos: 124, 125 and 126; SEQ ID Nos: 67, 68and 69/SEQ ID Nos: 127, 128 and 129; SEQ ID Nos: 67, 68 and 69/SEQ IDNos: 130, 131 and 132; SEQ ID Nos: 133, 134 and 135/SEQ ID Nos: 136, 137and 138; SEQ ID Nos: 139, 140 and 141/SEQ ID Nos: 142, 143 and 144; SEQID Nos: 55, 56 and 57/SEQ ID Nos: 145, 146 and 147; SEQ ID Nos: 148, 149and 150/SEQ ID Nos: 151, 152 and 153; SEQ ID Nos: 67, 68 and 69/SEQ IDNos: 154, 155 and 156; SEQ ID Nos: 157, 158 and 159/SEQ ID Nos: 160, 161and 162; SEQ ID Nos: 163, 164 and 165/SEQ ID Nos: 166, 167 and 168; SEQID Nos: 67, 68 and 69/SEQ ID Nos: 169, 170 and 171; SEQ ID Nos: 172, 173and 174/SEQ ID Nos: 175, 176 and 177; SEQ ID Nos: 178, 179 and 180/SEQID Nos: 181, 182 and 183; SEQ ID Nos: 184, 185 and 186/SEQ ID Nos: 187,188 and 189; SEQ ID Nos: 67, 68 and 69/SEQ ID Nos: 190, 191 and 192; SEQID Nos: 195, 196 and 197/SEQ ID Nos: 198, 199 and 200; SEQ ID Nos: 203,204 and 205/SEQ ID Nos: 206, 207 and 208; SEQ ID Nos: 61, 62 and 63/SEQID Nos: 210, 211 and 212; SEQ ID Nos: 67, 68 and 69/SEQ ID Nos: 214, 215and 216.

In another aspect, the invention features an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising an amino acid sequence that is at least 95% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201; and comprising a light chain variable domaincomprising an amino acid sequence that is at least 95% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In another aspect, the invention features an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising an amino acid sequence that is at least 96% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201; and comprising a light chain variable domaincomprising an amino acid sequence that is at least 96% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In another aspect, the invention features an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising an amino acid sequence that is at least 97% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201; and comprising a light chain variable domaincomprising an amino acid sequence that is at least 97% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In another aspect, the invention features an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising an amino acid sequence that is at least 98% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201; and comprising a light chain variable domaincomprising an amino acid sequence that is at least 98% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In another aspect, the invention features an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising an amino acid sequence that is at least 99% identicalto an amino acid sequence selected from the group consisting of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201; and comprising a light chain variable domaincomprising an amino acid sequence that is at least 99% identical to anamino acid sequence selected from the group consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In another aspect, the invention features an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain variabledomain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201; and comprising a lightchain variable domain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ IDNO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23,SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO.30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209and SEQ ID NO. 213.

In one embodiment of any one of the above aspects or embodiments, theantibody, or antigen-binding fragment thereof, has a K_(D) of at least1×10⁻⁶ M.

In another embodiment of any one of the above aspects or embodiments,the antibody, or antigen-binding fragment thereof, is an isolated humanantibody.

In one embodiment of any one of the above aspects or embodiments, theanti-OX40 antibody is an IgG. In a further embodiment, the antibody isan IgG1, IgG2, IgG3 or an IgG4 isotype.

In one embodiment of any one of the above aspects or embodiments, theantigen-binding fragment is a Fab fragment or an scFv.

In another embodiment, the invention features a pharmaceuticalcomposition comprising the anti-OX40 antibody, or antibody fragmentthereof, of any one of the above aspects or embodiments, and apharmaceutically acceptable carrier.

In one embodiment, the invention features a method for treating asubject having cancer, the method comprising administering an effectiveamount of the anti-OX40 antibody, or antigen-binding fragment thereof,of any one of the above aspects or embodiments to the subject. In oneembodiment, the cancer is selected from the group consisting of prostatecancer, breast cancer, ovarian cancer, head and neck cancer, bladdercancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer,leiomyoma, leiomyosarcoma, glioma, glioblastoma, esophageal cancer,liver cancer, kidney cancer, stomach cancer, colon cancer, cervicalcancer, uterine cancer, liver cancer and a hematological cancer. Inanother further embodiment, the cancer is selected from the groupconsisting of B chronic lymphocytic leukemia (B-CLL), B and T acutelymphocytic leukemia (ALL), acute myeloid leukemia (AML), chroniclymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairycell leukemia (HCL), myeloproliferative disorder/neoplasm (MPDS),myelodysplasia syndrome, non-Hodgkin's lymphoma (NHL), includingBurkitt's lymphoma (BL), Waldenstrom's Macroglobulinemia, mantle celllymphoma, AIDS-related lymphoma, Hodgkin's Lymphoma (HL), T celllymphoma (TCL), multiple myeloma (MM), plasma cell myeloma, plamocytoma,giant cell myeloma, heavy-chain myeloma, and light chain or Bence-Jonesmyeloma.

In one embodiment, the invention features a method for treating asubject having an inflammatory disease, the method comprisingadministering an effective amount of the anti-OX40 antibody, orantigen-binding fragment thereof, of any one of the above aspects orembodiments to the subject.

In a further embodiment, the inflammatory disease is selected from thegroup consisting of allergy, COPD, autoimmune disease, rheumatoidarthritis, asthma, graft versus host disease, Crohn's disease,ulcerative colitis, type-1 diabetes, multiple sclerosis, Systemic lupuserythematosis, lupus nephritis, Myasthenia Gravis, Grave's disease,transplant rejection, Wegener's granulomatosis, Henoch-Schonleinpurpura, systemic sclerosis, and viral-induced lung inflammation.

In one embodiment, the invention features a method for treating asubject having an infection, the method comprising administering aneffective amount of the anti-OX40 antibody, or antigen-binding fragmentthereof, of any one of the above aspects or embodiments to the subject.

In one embodiment, the invention features a method for treating adisease requiring either stimulation of immune responses or suppression,the method comprising administering an effective amount of the anti-OX40antibody, or antigen-binding fragment thereof, of any one of the aboveaspects or embodiments to the subject. In a further embodiment, thedisease is selected from the group consisting of cancer, an inflammatorydisease, and a viral infection.

Also included in the invention are nucleic acids encoding the amino acidsequences disclosed herein, as well as methods of making the antibodiesand fragments of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph that shows the binding of anti-OX40 antibody clones onactivated human T-cells measured by the percent CD3+OX40+ T cells. Theanti-OX40 clones that were tested are shown on the x-axis. Secondaryantibody alone was used as a negative control. A commercially availableAPC-labelled anti-human OX40 (clone Ber-ACT35) was used as positivecontrol.

FIG. 2A is a graph that shows the luciferase activity measured byrelative light units (RLUs) for the control experimental conditionsafter 6 hours of stimulation. As shown in FIG. 2A, unstimulated cells,isotype control FeH3 antibody (Ctrl FeH3) and polyclonal anti-OX40antibody did not show an increase in RLUs, indicating that the NFkBpathway was not activated. The positive controls, solubleanti-Histidine+purified rhOX40L (rhOX40L+αHis) and soluble TNFα, showedhigher RLUs, indicating an increase in NFkB activity.

FIG. 2B is a graph that shows luciferase activity measured by RLUs forthe anti-OX40 antibody clones tested after 6 hours of stimulation.Unstimulated cells and isotype control FeH3 antibody (Ctrl FeH3) areshown as negative controls. Soluble anti-Histidine+purified rhOX40L(rhOX40L+αHis) is shown as a positive control. The line bisecting thegraph shows the negative control RLU levels as a reference. As shown inFIG. 2B, all of the OX40 clones showed an increase in RLUs over thenegative control values. Clones 1C4, 4D7 1D9 and 1B2 showed thestrongest NFkB activation (>50% of signal observed with purifiedrhOX40L+anti-His).

FIG. 3A is a graph that shows the luciferase activity measured byrelative light units (RLUs) for the control experimental conditionsafter ˜15 hours of stimulation. As shown in FIG. 3A, unstimulated cells,isotype control FeH3 antibody (Ctrl FeH3) did not show an increase inRLUs, indicating that the NFkB pathway was not activated. The positivecontrols, soluble anti-Histidine+purified rhOX40L (rhOX40L+αHis) andsoluble TNFα, showed higher RLUs, indicating an increase in NFkBactivity.

FIG. 3B is a graph that shows luciferase activity measured by RLUs forthe anti-OX40 antibody clones tested after ˜15 hours of stimulation.Unstimulated cells and isotype control FeH3 antibody (Ctrl FeH3) areshown as negative controls. Soluble anti-Histidine+purified rhOX40L(rhOX40L+αHis) is shown as a positive control. The line bisecting thegraph shows the negative control RLU levels as a reference. As shown inFIG. 3B, most of the OX40 clones showed an increase in RLUs over thenegative control values. In particular clones 1A11, 1C4, 4D7, 1D9, 5C11,2B4, 4D9, 3C10 and 1B2 were among the most active.

FIG. 4A is a graph that shows the percentage of CD3+CD25+ activated Tcells as measured by flow cytometry. Anti-CD3 alone and FeD2, an isotypecontrol antibody, were used as negative controls. Anti-CD28 was used asa positive control. The line bisecting the graph shows the negativecontrol levels of percentage of CD3+CD25+ activated T cells as areference.

FIG. 4B is a graph that shows the percentage of CD3+CD25+ activated Tcells as measured by flow cytometry. Anti-CD3 alone and FeD2, an isotypecontrol antibody, were used as negative controls. Anti-CD28 was used asa positive control. The line bisecting the graph shows the negativecontrol levels of percentage of CD3+CD25+ activated T cells as areference.

As shown in FIG. 4A and FIG. 4B, a number of anti-OX40 clones showedsignificant T cell co-stimulation. Among those clones showing T cellco-stimulation, clones 2B4, 4D7, 2B3, 4G9 and 1B1 showed the most robustagonist activity.

DETAILED DESCRIPTION

Definitions

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a confirmation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding fragment of an antibody), antibodyderivatives, and antibody analogs. The antigen binding protein cancomprise, for example, an alternative protein scaffold or artificialscaffold with grafted CDRs or CDR derivatives. Such scaffolds include,but are not limited to, antibody-derived scaffolds comprising mutationsintroduced to, for example, stabilize the three-dimensional structure ofthe antigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

The term “antibody” is synonymous with immunoglobulin and is to beunderstood as commonly known in the art. The basic antibody structuralunit is a tetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kDa) and one“heavy” chain (about 50-70 kDa). Generally, the amino-terminal portionof each antibody chain includes a variable region that is primarilyresponsible for antigen recognition. The carboxy-terminal portion ofeach chain defines a constant region, e.g., responsible for effectorfunction. Human light chains are classified as kappa or lambda lightchains. Heavy chains are classified as mu, delta, gamma, alpha, orepsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, andIgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 3 ormore amino acids.

The variable regions of each heavy/light chain pair (VH/VL),respectively, form the antigen binding site. The variable regions ofantibody heavy and light chains (VH/VL) exhibit the same generalstructure of relatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regionsor CDRs. From N-terminus to C-terminus, both light and heavy chainscomprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Theassignment of amino acids to each domain is known in the art, including,for example, definitions as described in Kabat et al. in Sequences ofProteins of Immunological Interest, 5^(th) Ed., US Dept. of Health andHuman Services, PHS, NIH, NIH Publication no. 91-3242, 1991 (hereinreferred to as “Kabat numbering”). For example, the CDR regions of anantibody can be determined according to Kabat numbering.

The terms “intact antibody” or “full length antibody” refer to anantibody composed of two identical antibody light chains and twoidentical antibody heavy chains that each contain an Fc region.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein). Generally the variable regions, particularlythe CDRs, of an antibody interact with the epitope.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

The terms “anti-OX40 antibody” and “an antibody that binds to OX40”refer to an antibody that is capable of binding OX40 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting OX40, including human OX40.

The term “monospecific”, as used herein, refers to an antibody, orantigen binding fragment thereof, that displays an affinity for oneparticular epitope. In contrast, a bispecific antibody, orantigen-binding fragment thereof, displays affinity for two differentepitopes. In one embodiment, the methods and compositions describedherein are useful for intracellular delivery of a monospecific antibody,or antigen-binding fragment thereof. In one embodiment, the anti-OX40antibody, or antigen-binding fragment thereof, of the invention ismonospecific.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bispecific antibody” which recognizes two distinct epitopes on thesame or different antigens.

The terms “specific binding”, “specifically binds” or “specificallybinding”, as used herein in the context of an antibody, refer tonon-covalent or covalent preferential binding of an antibody to anantigen relative to other molecules or moieties (e.g., an antibodyspecifically binds to a particular antigen relative to other availableantigens). In one embodiment, an antibody specifically binds to anantigen (e.g., OX40) if it binds to the antigen with a dissociationconstant K_(D) of 10⁻⁵ M or less (e.g., 10⁻⁶ M or less, 10⁻⁷ M or less,10⁻⁸ M or less, 10⁻⁹ M or less, or 10⁻¹⁰ M or less).

The term “human antibody”, as used herein, refers to an antibody, or anantigen binding fragment of an antibody, comprising heavy and lightschains derived from human immunoglobulin sequences. Human antibodies maybe identified in a variety of ways, examples of which are describedbelow, including through the immunization with an antigen of interest ofa mouse that is genetically modified to express antibodies derived fromhuman heavy and/or light chain-encoding genes. In one embodiment, ahuman antibody is made using recombinant methods such that theglycosylation pattern of the antibody is different than an antibodyhaving the same sequence if it were to exist in nature.

The term “chimeric antibody” refers to an antibody that contains one ormore regions derived from a particular source or species, and one ormore regions derived from a different source or species.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like,” i.e., more similar tohuman germline variable sequences. A “humanized antibody” is an antibodyor a variant, derivative, analog, or fragment thereof, whichimmunospecifically binds to an antigen of interest and which comprises aframework (FR) region having substantially the amino acid sequence of ahuman antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody.

A “humanized antibody” refers to an antibody having a sequence thatdiffers from the sequence of an antibody derived from a non-humanspecies by one or more amino acid substitutions, deletions, and/oradditions. Generally, a humanized antibody is less likely to induce animmune response, and/or induces a less severe immune response, ascompared to the non-human species antibody, e.g., a murine or chimericantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. Examples of how to make humanizedantibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and5,877,293.

An “antibody fragment”, “antibody portion”, “antigen-binding fragment ofan antibody”, or “antigen-binding portion of an antibody” refers to amolecule other than an intact antibody that comprises a portion of anintact antibody that binds the antigen to which the intact antibodybinds. Examples of antibody fragments include, but are not limited to,Fv, Fab, Fab′, Fab′-SH, F(ab′)₂; Fd; and Fv fragments, as well as dAb;diabodies; linear antibodies; single-chain antibody molecules (e.g.scFv); polypeptides that contain at least a portion of an antibody thatis sufficient to confer specific antigen binding to the polypeptide.Antigen binding portions of an antibody may be produced by recombinantDNA techniques or by enzymatic or chemical cleavage of intactantibodies. Antigen binding portions include, inter alia, Fab, Fab′,F(ab′)2, Fv, domain antibodies (dAbs), and complementarity determiningregion (CDR) fragments, chimeric antibodies, diabodies, triabodies,tetrabodies, and

In one embodiment, the antibody fragment is an scFv. A single-chainantibody (scFv) is an antibody in which a V_(L) and a V_(H) region arejoined via a linker (e.g., a synthetic sequence of amino acid residues)to form a continuous protein chain (see e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883)).

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or V_(L) domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US App Pub20/0202512; 2004/0202995; 2004/0038291; 2004/0009507; 2003/0039958, andWard et al., Nature 341:544-546, 1989).

Diabodies are bivalent antibodies comprising two polypeptide chains,wherein each polypeptide chain comprises VH and VL domains joined by alinker that is too short to allow for pairing between two domains on thesame chain, thus allowing each domain to pair with a complementarydomain on another polypeptide chain (see, e.g., Holliger et al., 1993,Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljak et al., 1994,Structure 2:1121-23). If the two polypeptide chains of a diabody areidentical, then a diabody resulting from their pairing will have twoidentical antigen binding sites. Polypeptide chains having differentsequences can be used to make a diabody with two different antigenbinding sites. Similarly, tribodies and tetrabodies are antibodiescomprising three and four polypeptide chains, respectively, and formingthree and four antigen binding sites, respectively, which can be thesame or different.

The “percent identity” or “percent homology” of two polynucleotide ortwo polypeptide sequences is determined by comparing the sequences usingthe GAP computer program (a part of the GCG Wisconsin Package, version10.3 (Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragmentthereof, as described herein.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293, 293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. In one embodiment, ahost cell is a mammalian host cell, but is not a human host cell.Typically, a host cell is a cultured cell that can be transformed ortransfected with a polypeptide-encoding nucleic acid, which can then beexpressed in the host cell. The phrase “recombinant host cell” can beused to denote a host cell that has been transformed or transfected witha nucleic acid to be expressed. A host cell also can be a cell thatcomprises the nucleic acid but does not express it at a desired levelunless a regulatory sequence is introduced into the host cell such thatit becomes operably linked with the nucleic acid. It is understood thatthe term host cell refers not only to the particular subject cell butalso to the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

The term “recombinant antibody” refers to an antibody that is expressedfrom a cell (or cell line) transfected with an expression vector (orpossibly more than one expression vector) comprising the coding sequenceof the antibody, or a portion thereof (e.g., a DNA sequence encoding aheavy chain or a light chain variable region as described herein). Inone embodiment, said coding sequence is not naturally associated withthe cell. In one embodiment, a recombinant antibody has a glycosylationpattern that is different than the glycosylation pattern of an antibodyhaving the same sequence if it were to exist in nature. In oneembodiment, a recombinant antibody is expressed in a mammalian host cellwhich is not a human host cell. Notably, individual mammalian host cellshave unique glycosylation patterns.

The term “effective amount” as used herein, refers to that amount of anantibody, or an antigen binding portion thereof that binds OX40, whichis sufficient to effect treatment of a disease associated with OX40signaling, as described herein, when administered to a subject.Therapeutically effective amounts of antibodies provided herein, whenused alone or in combination, will vary depending upon the relativeactivity of the antibodies and combinations (e.g., in inhibiting cellgrowth) and depending upon the subject and disease condition beingtreated, the weight and age of the subject, the severity of the diseasecondition, the manner of administration and the like, which can readilybe determined by one of ordinary skill in the art.

The term “isolated” refers to a protein (e.g., an antibody) that issubstantially free of other cellular material. In one embodiment, anisolated antibody is substantially free of other proteins from the samespecies. In one embodiment, an isolated antibody is expressed by a cellfrom a different species and is substantially free of other proteinsfrom the different species. A protein may be rendered substantially freeof naturally associated components (or components associated with thecellular expression system used to produce the antibody) by isolation,using protein purification techniques well known in the art. In oneembodiment, the antibodies, or antigen binding fragments, of theinvention are isolated.

As used herein, the phrase “OX40 activation” refers to activation of theOX40 receptor. Generally, OX40 activation results in signaltransduction.

The term “OX40,” as used herein, refers to any native OX40 from anyvertebrate source, including mammals such as primates (e.g., humans) androdents (e.g., mice and rats), unless otherwise indicated. The termencompasses “full-length,” unprocessed OX40 as well as any form of OX40that results from processing in the cell. The term also encompassesnaturally occurring variants of OX40, for example, splice variants orallelic variants. The amino acid sequence of an exemplary human OX40lacking the signal peptide is shown in SEQ ID NO. 217(LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI). In one embodiment, the antibody, or fragment, ofthe invention binds to OX40 as set forth in SEQ ID NO: 217.

OX40 Antigen Binding Proteins

The present invention pertains to OX40 binding proteins, particularlyanti-OX40 antibodies, or antigen-binding portions thereof, and usesthereof. Various aspects of the invention relate to antibodies andantibody fragments, pharmaceutical compositions, nucleic acids,recombinant expression vectors, and host cells for making suchantibodies and fragments. Methods of using the antibodies of theinvention to detect human OX40, to stimulate OX40 activity, either invitro or in vivo, and to prevent or treat disorders such as cancer arealso encompassed by the invention.

As described in Table 1 below, included in the invention are novel humanantibody heavy and light chain variable regions and CDRs that arespecific to human OX40.

In one embodiment, the invention provides an anti-OX40 antibody, or anantigen-binding fragment thereof, that comprises a heavy chain having avariable domain comprising an amino acid sequence as set forth in anyone of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ IDNO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34,SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO.46, SEQ ID NO. 193 and SEQ ID NO. 201. In one embodiment, the inventionprovides an anti-OX40 antibody, or an antigen-binding fragment thereof,that comprises a light chain having a variable domain comprising anamino acid sequence as set forth in any one of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ IDNO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ IDNO. 202, SEQ ID NO. 209 and SEQ ID NO. 213. In one embodiment, theinvention provides an anti-OX40 antibody, or an antigen-binding fragmentthereof, that comprises a light chain having a variable domaincomprising an amino acid sequence as set forth in any one of SEQ ID NO.2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO.12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ IDNO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33,SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO.41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ IDNO. 194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213; and a heavychain having a variable domain comprising an amino acid sequence as setforth in any one of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201.

In one embodiment, the present disclosure provides a human antibody ofan IgG class that binds to a human OX40 epitope with a binding affinityof at least 10⁻⁶M, where the antibody, or antigen-binding fragment, hasa heavy chain variable domain sequence which is at least 95% identical,at least 96% identical, at least 97% identical, at least 98% identical,or at least 99% identical, or identical, to an amino acid sequenceselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ IDNO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42,SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, and hasa light chain variable domain sequence that is at least 95% identical,at least 96% identical, at least 97% identical, at least 98% identical,or at least 99% identical, or identical to an amino acid sequenceselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ IDNO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202,SEQ ID NO. 209 and SEQ ID NO. 213.

In one embodiment, the invention features an isolated anti-hOX40 humanantibody comprising a heavy chain/light chain variable domain amino acidsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called OX1A11 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called OX1B1 herein),SEQ ID NO. 5/SEQ ID NO. 6 (called OX1B2 herein), SEQ ID NO. 7/SEQ ID NO.8 (called OX1B3 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called OX1C4herein), SEQ ID NO. 11/SEQ ID NO. 12 (called OX1C5 herein), SEQ ID NO.13/SEQ ID NO. 14 (called OX1D9 herein), SEQ ID NO. 15/SEQ ID NO. 16(called OX1E10 herein), SEQ ID NO. 7/SEQ ID NO. 17 (called OX1E7herein), SEQ ID NO. 3/SEQ ID NO. 18 (called OX1F2 herein), SEQ ID NO.19/SEQ ID NO. 20 (called OX1G9), SEQ ID NO. 21/SEQ ID NO. 22 (calledOX2B12 herein), SEQ ID NO. 7/SEQ ID NO. 23 (called OX2B3 herein), SEQ IDNO. 24/SEQ ID NO. 25 (called OX2B4 herein), SEQ ID NO. 7/SEQ ID NO. 26(called OX2B6 herein), SEQ ID NO. 7/SEQ ID NO. 27 (called OX2F2 herein),SEQ ID NO. 7/SEQ ID NO. 28 (called OX2G2 herein), SEQ ID NO. 29/SEQ IDNO. 30 (called OX3C10 herein), SEQ ID NO. 31/SEQ ID NO. 32 (calledOX4A11 herein), SEQ ID NO. 3/SEQ ID NO. 33 (called OX4A12 herein), SEQID NO. 34/SEQ ID NO. 35 (called OX4B6 herein), SEQ ID NO. 7/SEQ ID NO.36 (called OX4D4 herein), SEQ ID NO. 37/SEQ ID NO. 38 (called OX4D7herein), SEQ ID NO. 39/SEQ ID NO. 40 (called OX4D9 herein), SEQ ID NO.7/SEQ ID NO. 41 (called OX4G9 herein), SEQ ID NO. 42/SEQ ID NO. 43(called OX4H4 herein), SEQ ID NO. 44/SEQ ID NO. 45 (called OX5B9herein), SEQ ID NO. 46/SEQ ID NO. 47 (called OX5C1 herein), SEQ ID NO.7/SEQ ID NO. 48 (called OX5D7 herein), SEQ ID NO. 193/SEQ ID NO. 194(called OX4B5 herein), SEQ ID NO. 201/SEQ ID NO. 202 (called OX2E5herein), SEQ ID NO. 5/SEQ ID NO. 209 (called OX2B5 herein), and SEQ IDNO. 7/SEQ ID NO. 213 (called OX5C11 herein).

Complementarity determining regions (CDRs) are known as hypervariableregions both in the light chain and the heavy chain variable domains ofan antibody. The more highly conserved portions of variable domains arecalled the framework (FR). Complementarity determining regions (CDRs)and framework regions (FR) of a given antibody may be identified usingsystems known in the art, such as those described by Kabat et al. supra;Lefranc et al., supra and/or Honegger and Pluckthun, supra. For example,the numbering system described in Kabat et al. (1991, NIH Publication91-3242, National Technical Information Service, Springfield, Va.) iswell known to those in the art. Kabat et al. defined a numbering systemfor variable domain sequences that is applicable to any antibody. One ofordinary skill in the art can unambiguously assign this system of “Kabatnumbering” to any variable domain amino acid sequence, without relianceon any experimental data beyond the sequence itself.

In certain embodiments, the present invention provides an anti-OX40antibody comprising the CDRs of the heavy and light chain variabledomains described in Table 1 (SEQ ID Nos: 1 to 212). For example, theinvention provides an anti-OX40 antibody, or antigen-binding fragmentthereof, comprising a heavy chain variable region having the CDRsdescribed in an amino acid sequence as set forth in any one of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201. In one embodiment, the invention provides ananti-OX40 antibody, or antigen-binding fragment thereof, comprising alight chain variable region having the CDRs described in an amino acidsequence as set forth in any one of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ IDNO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202,SEQ ID NO. 209 and SEQ ID NO. 213. In one embodiment, the inventionprovides an anti-OX40 antibody, or antigen-binding fragment thereof,comprising a light chain variable region having the CDRs described in anamino acid sequence as set forth in any one of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ IDNO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ IDNO. 202, SEQ ID NO. 209 and SEQ ID NO. 213; and a heavy chain variableregion having the CDRs described in an amino acid sequence as set forthin any one of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201.

In one embodiment, the present invention features an isolated humananti-human OX40 (hOX40) antibody, or an antigen-binding fragmentthereof, comprising a heavy chain variable domain comprising a heavychain CDR set (CDR1, CDR2, and CDR3) selected from the group consistingof SEQ ID Nos: 49, 50 and 51; SEQ ID Nos: 55, 56 and 57; SEQ ID Nos: 61,62 and 63; SEQ ID Nos: 67, 68, and 69; SEQ ID Nos: 73, 74, and 75; SEQID Nos: 79, 80, and 81; SEQ ID Nos: 85, 86, and 87; SEQ ID Nos: 91, 92,and 93; SEQ ID Nos: 103, 104 and 105; SEQ ID Nos: 109, 110 and 111; SEQID Nos: 118, 119 and 120; SEQ ID Nos: 133, 134 and 135; SEQ ID Nos: 139,140, 141; SEQ ID Nos: 148, 149 and 150; SEQ ID Nos: 157, 158 and 159;SEQ ID Nos: 163, 164 and 165; SEQ ID Nos: 172, 173 and 174; SEQ ID Nos:178, 179 and 180; SEQ ID Nos: 184, 185 and 186; SEQ ID Nos. 195, 196 and197; and SEQ ID Nos. 203, 204 and 205; and a light chain variable domaincomprising a light chain CDR set (CDR1, CDR2, and CDR3) selected fromthe group consisting of SEQ ID Nos: 52, 53 and 54; SEQ ID Nos: 58, 59and 60; SEQ ID Nos: 64, 65 and 66; SEQ ID Nos: 70, 71 and 72; SEQ IDNos: 76, 77 and 78; SEQ ID Nos: 82, 83 and 84; SEQ ID Nos: 88, 89 and90; SEQ ID Nos: 94, 95 and 96; SEQ ID Nos: 97, 98 and 99; SEQ ID Nos:100, 101 and 102; SEQ ID Nos: 106, 107 and 108; SEQ ID Nos: 112, 113 and114; SEQ ID Nos: 115, 116 and 117; SEQ ID Nos: 121, 122 and 123; SEQ IDNos: 124, 125 and 126; SEQ ID Nos: 127, 128 and 129; SEQ ID Nos: 130,131 and 132; SEQ ID Nos: 136, 137 and 138; SEQ ID Nos: 142, 143 and 144;SEQ ID Nos: 145, 146 and 147; SEQ ID Nos: 151, 152 and 153; SEQ ID Nos:154, 155 and 156; SEQ ID Nos: 160, 161 and 162; SEQ ID Nos: 166, 167 and168; SEQ ID Nos: 169, 170 and 171; SEQ ID Nos: 175, 176 and 177; SEQ IDNos: 181, 182 and 183; SEQ ID Nos: 187, 188 and 189; SEQ ID Nos: 190,191 and 192; SEQ ID Nos. 198, 199 and 200; SEQ ID Nos. 206, 207 and 208;SEQ ID Nos. 210, 211 and 212; and SEQ ID Nos. 214, 215 and 216.

In one embodiment, the antibody of the invention comprises a heavy chainCDR set/light chain CDR set selected from the group consisting of theheavy chain variable domain CDR set of SEQ ID Nos: 49, 50 and 51/thelight chain variable domain CDR set of 52, 53 and 54; the heavy chainvariable domain CDR set of SEQ ID Nos: 55, 56 and 57/the light chainvariable domain CDR set of 58, 59 and 60; the heavy chain variabledomain CDR set of SEQ ID Nos: 61, 62, and 63/the light chain variabledomain CDR set of 64, 65 and 66; the heavy chain variable domain CDR setof SEQ ID Nos: 67, 68 and 69/the light chain variable domain CDR set of70, 71 and 72; the heavy chain variable domain CDR set of SEQ ID Nos:73, 74 and 75/the light chain variable domain CDR set of 76, 77 and 78;the heavy chain variable domain CDR set of SEQ ID Nos: 79, 80 and 81/thelight chain variable domain CDR set of 82, 83 and 84; the heavy chainvariable domain CDR set of SEQ ID Nos: 85, 86 and 87/the light chainvariable domain CDR set of 88, 89 and 90; the heavy chain variabledomain CDR set of SEQ ID Nos: 91, 92 and 93/the light chain variabledomain CDR set of 94, 95 and 96; the heavy chain variable domain CDR setof SEQ ID Nos: 67, 68 and 69/the light chain variable domain CDR set of97, 98 and 99; the heavy chain variable domain CDR set of SEQ ID Nos:55, 56 and 57/the light chain variable domain CDR set of 100, 101 and102; the heavy chain variable domain CDR set of SEQ ID Nos: 103, 104 and105/the light chain variable domain CDR set of 106, 107 and 108; theheavy chain variable domain CDR set of SEQ ID Nos: 109, 110 and 111/thelight chain variable domain CDR set of 112, 113 and 114; the heavy chainvariable domain CDR set of SEQ ID Nos: 67, 68 and 69/the light chainvariable domain CDR set of 115, 116 and 117; the heavy chain variabledomain CDR set of SEQ ID Nos: 118, 119 and 120/the light chain variabledomain CDR set of 121, 122 and 123; the heavy chain variable domain CDRset of SEQ ID Nos: 67, 68 and 69/the light chain variable domain CDR setof 124, 125 and 126; the heavy chain variable domain CDR set of SEQ IDNos: 67, 68 and 69/the light chain variable domain CDR set of 127, 128and 129; the heavy chain variable domain CDR set of SEQ ID Nos: 67, 68and 69/the light chain variable domain CDR set of 130, 131 and 132; theheavy chain variable domain CDR set of SEQ ID Nos: 133, 134 and 135/thelight chain variable domain CDR set of 136, 137 and 138; the heavy chainvariable domain CDR set of SEQ ID Nos: 139, 140 and 141/the light chainvariable domain CDR set of 142, 143 and 144; the heavy chain variabledomain CDR set of SEQ ID Nos: 55, 56 and 57/the light chain variabledomain CDR set of 145, 146 and 147; the heavy chain variable domain CDRset of SEQ ID Nos: 148, 149 and 150/the light chain variable domain CDRset of 151, 152 and 153; the heavy chain variable domain CDR set of SEQID Nos: 67, 68 and 69/the light chain variable domain CDR set of 154,155 and 156; the heavy chain variable domain CDR set of SEQ ID Nos: 157,158 and 159/the light chain variable domain CDR set of 160, 161 and 162;the heavy chain variable domain CDR set of SEQ ID Nos: 163, 164 and165/the light chain variable domain CDR set of 166, 167 and 168; theheavy chain variable domain CDR set of SEQ ID Nos: 67, 68 and 69/thelight chain variable domain CDR set of 169, 170, 171; the heavy chainvariable domain CDR set of SEQ ID Nos: 172, 173 and 174/the light chainvariable domain CDR set of 175, 176 and 177; the heavy chain variabledomain CDR set of SEQ ID Nos: 178, 179 and 180/the light chain variabledomain CDR set of 181, 182 and 183; the heavy chain variable domain CDRset of SEQ ID Nos: 184, 185 and 186/the light chain variable domain CDRset of 187, 188 and 189; the heavy chain variable domain CDR set of SEQID Nos: 67, 68 and 69/the light chain variable domain CDR set of 190,191 and 192; the heavy chain variable domain CDR set of SEQ ID Nos: 195,196 and 197/the light chain variable domain CDR set of 198, 199 and 200;the heavy chain variable domain CDR set of SEQ ID Nos: 203, 204 and205/the light chain variable domain CDR set of 206, 207 and 208; theheavy chain variable domain CDR set of SEQ ID Nos: 61, 62 and 63/thelight chain variable domain CDR set of 210, 211 and 212; and the heavychain variable domain CDR set of SEQ ID Nos: 67, 68 and 69/the lightchain variable domain CDR set of 214, 215 and 216.

In one embodiment, the invention provides an anti-OX40 antibody, or anantigen-binding fragment thereof, comprising a heavy chain comprising aCDR3 domain as set forth in any one of SEQ ID NO. 1, SEQ ID NO. 3, SEQID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29,SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO.42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, andcomprising a variable domain comprising an amino acid sequence that hasat least 95%, at least 96%, at least 97%, at least 98%, at least 99%, orat least 100% identity to a sequence as set forth in any one of SEQ IDNO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37,SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO.193 and SEQ ID NO. 201. In one embodiment, the invention provides ananti-OX40 antibody, or an antigen-binding fragment thereof, comprising alight chain comprising a CDR3 domain as set forth in any one of SEQ IDNO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26,SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213, andhaving a light chain variable domain comprising an amino acid sequencethat has at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or at least 100% identity to a sequence as set forth in anyone of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ IDNO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25,SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO.32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ IDNO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO.213. Thus, in certain embodiments, the CDR3 domain is held constant,while variability may be introduced into the remaining CDRs and/orframework regions of the heavy and/or light chains, while the antibody,or antigen binding fragment thereof, retains the ability to bind to OX40and retains the functional characteristics, e.g., binding affinityand/or the ability to activate T cells, of the parent.

One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an antigen binding protein.

An antigen binding protein may incorporate the CDR(s) as part of alarger polypeptide chain, may covalently link the CDR(s) to anotherpolypeptide chain, or may incorporate the CDR(s) noncovalently. The CDRspermit the antigen binding protein to specifically bind to a particularantigen of interest.

In one embodiment, the substitutions made within a heavy or light chainthat is at least 95% identical (or at least 96% identical, or at least97% identical, or at least 98% identical, or at least 99% identical) areconservative amino acid substitutions. A “conservative amino acidsubstitution” is one in which an amino acid residue is substituted byanother amino acid residue having a side chain (R group) with similarchemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change thefunctional properties of a protein. In cases where two or more aminoacid sequences differ from each other by conservative substitutions, thepercent sequence identity or degree of similarity may be adjustedupwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in theart. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, hereinincorporated by reference. Examples of groups of amino acids that haveside chains with similar chemical properties include (1) aliphatic sidechains: glycine, alanine, valine, leucine and isoleucine; (2)aliphatic-hydroxyl side chains: serine and threonine; (3)amide-containing side chains: asparagine and glutamine; (4) aromaticside chains: phenylalanine, tyrosine, and tryptophan; (5) basic sidechains: lysine, arginine, and histidine; (6) acidic side chains:aspartate and glutamate, and (7) sulfur-containing side chains arecysteine and methionine.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having the antigen binding regionsof any of the antibodies described in Table 1.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX1A11. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 1, and a light chain variabledomain sequence as set forth in SEQ ID NO: 2. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 1, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 2. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 1, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 2. In one embodiment, the invention features an anti-OX40antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 51, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 50, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 49; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 54, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 53, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 52. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX2E5. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 201, and a light chainvariable domain sequence as set forth in SEQ ID NO: 202. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 201, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 202. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 201, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 202. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 205, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 204, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 203; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 208, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:207, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 206. The antibody may further be an IgG1 or an IgG4isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX2B12. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 21, and a light chainvariable domain sequence as set forth in SEQ ID NO: 22. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 21, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 22. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 21, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 22. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 111, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 110, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 109; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 114, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:113, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 112. The antibody may further be an IgG1 or an IgG4isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX1B1. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 3, and a light chain variabledomain sequence as set forth in SEQ ID NO: 4. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 3, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 4. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 3, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 4. In one embodiment, the invention features an anti-OX40antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 57, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 56, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 55; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 60, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 59, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 58. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX5C1. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 46, and a light chainvariable domain sequence as set forth in SEQ ID NO: 47. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 46, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 47. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 46, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 47. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 186, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 185, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 184; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 189, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:188, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 187. The antibody may further be an IgG1 or an IgG4isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX4D7. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 37, and a light chainvariable domain sequence as set forth in SEQ ID NO: 38. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 37, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 38. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 37, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 38. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 159, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 158, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 157; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 162, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:161, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 160. The antibody may further be an IgG1 or an IgG4isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX5B9. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 44, and a light chainvariable domain sequence as set forth in SEQ ID NO: 45. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 44, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 45. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 44, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 45. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 180, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 179, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 178; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 183, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:182, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 181. The antibody may further be an IgG1 or an IgG4isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX1C4. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 9, and a light chain variabledomain sequence as set forth in SEQ ID NO: 10. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 9, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 10. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 9, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 10. In one embodiment, the invention features an anti-OX40antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 75, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 74, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 73; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 78, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 77, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 76. Theantibody may further be an IgG1 or an IgG4 isotype.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX4B5. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 193, and a light chainvariable domain sequence as set forth in SEQ ID NO: 194. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 193, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 194. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 193, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 194. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 197, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 195, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 195; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 200, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:199, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 198.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX2B5. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 5, and a light chain variabledomain sequence as set forth in SEQ ID NO: 209. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 5, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 209. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 5, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 209. In one embodiment, the invention features ananti-OX40 antibody, or an antigen-binding portion thereof, comprising aheavy chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 63, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 62, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 61; andcomprising a light chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 212, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 211, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 210.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX2B4. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 24, and a light chainvariable domain sequence as set forth in SEQ ID NO: 25. In oneembodiment, the invention is directed to an antibody having a heavychain variable domain comprising the CDRs of SEQ ID NO: 24, and a lightchain variable domain comprising the CDRs of SEQ ID NO: 25. In oneembodiment, the invention features an isolated human antibody, orantigen-binding fragment thereof, that comprises a heavy chain variableregion having an amino acid sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical to the sequence set forth in SEQ ID NO: 24, andcomprises a light chain variable region having an amino acid sequencethat is at least 95% identical, at least 96% identical, at least 97%identical, at least 98% identical, or at least 99% identical to thesequence set forth in SEQ ID NO: 25. In one embodiment, the inventionfeatures an anti-OX40 antibody, or an antigen-binding portion thereof,comprising a heavy chain variable region comprising a CDR3 domaincomprising the amino acid as set forth in SEQ ID NO: 120, a CDR2 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 119, and aCDR1 domain comprising the amino acid sequence as set forth in SEQ IDNO: 118; and comprising a light chain variable region comprising a CDR3domain comprising the amino acid as set forth in SEQ ID NO: 123, a CDR2domain comprising the amino acid sequence as set forth in SEQ ID NO:122, and a CDR1 domain comprising the amino acid sequence as set forthin SEQ ID NO: 121.

In one embodiment, the present invention is directed to an antibody, oran antigen binding fragment thereof, having antigen binding regions ofantibody OX2B3. In one embodiment, the invention provides an antibody,or antigen-binding fragment thereof, comprising a heavy chain variabledomain sequence as set forth in SEQ ID NO: 7, and a light chain variabledomain sequence as set forth in SEQ ID NO: 23. In one embodiment, theinvention is directed to an antibody having a heavy chain variabledomain comprising the CDRs of SEQ ID NO: 7, and a light chain variabledomain comprising the CDRs of SEQ ID NO: 23. In one embodiment, theinvention features an isolated human antibody, or antigen-bindingfragment thereof, that comprises a heavy chain variable region having anamino acid sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical to the sequence set forth in SEQ ID NO: 7, and comprises alight chain variable region having an amino acid sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical to the sequence set forthin SEQ ID NO: 23. In one embodiment, the invention features an anti-OX40antibody, or an antigen-binding portion thereof, comprising a heavychain variable region comprising a CDR3 domain comprising the amino acidas set forth in SEQ ID NO: 69, a CDR2 domain comprising the amino acidsequence as set forth in SEQ ID NO: 68, and a CDR1 domain comprising theamino acid sequence as set forth in SEQ ID NO: 67; and comprising alight chain variable region comprising a CDR3 domain comprising theamino acid as set forth in SEQ ID NO: 117, a CDR2 domain comprising theamino acid sequence as set forth in SEQ ID NO: 116, and a CDR1 domaincomprising the amino acid sequence as set forth in SEQ ID NO: 115.

The antibody of the invention may further be an IgG1 or an IgG4 isotype.

As described in Table 1, antibodies OX1B3, OX1E7, OX2B3, OX2B6, OX2F2,OX2G2, OX4D4, OX4G9, OX5D7 and OX5C11 have a heavy chain variable regionhaving an amino acid sequence as set forth in SEQ ID NO. 7. As alsodescribed in Table 1, antibodies OX1B1, OX1F2 and OX4A12 have a heavychain variable region having an amino acid sequence as set forth in SEQID NO. 3.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)2 fragments.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (VL and VH). The resulting polypeptides can fold back onthemselves to form antigen-binding monomers, or they can form multimers(e.g., dimers, trimers, or tetramers), depending on the length of aflexible linker between the two variable domains (Kortt et al., 1997,Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different VL and VH-comprising polypeptides, one can formmultimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

In certain embodiments, the present disclosure provides a Fab fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99%, or 100%identical, to the amino acid sequences selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, and that has a light chainvariable domain sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, at least 99%,or 100% identical to the amino acid sequence consisting of SEQ ID NO. 2,SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12,SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO.20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ IDNO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41,SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO.194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213. Preferably, thehuman antibody Fab fragment has both a heavy chain variable domainregion and a light chain variable domain region wherein the antibody hasa heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO. 7/SEQ ID NO. 17, SEQ ID NO.3/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO.22, SEQ ID NO. 7/SEQ ID NO. 23, SEQ ID NO. 24/SEQ ID NO. 25, SEQ ID NO.7/SEQ ID NO. 26, SEQ ID NO. 7/SEQ ID NO. 27, SEQ ID NO. 7/SEQ ID NO. 28,SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO.3/SEQ ID NO. 33, SEQ ID NO. 34/SEQ ID NO. 35, SEQ ID NO. 7/SEQ ID NO.36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO.7/SEQ ID NO. 41, SEQ ID NO. 42/SEQ ID NO. 43), SEQ ID NO. 44/SEQ ID NO.45, SEQ ID NO. 46/SEQ ID NO. 47 and SEQ ID NO. 7/SEQ ID NO. 48, SEQ IDNO. 193/SEQ ID NO. 194, SEQ ID NO. 201/SEQ ID NO. 202, SEQ ID NO. 5/SEQID NO. 209 and SEQ ID NO. 7/SEQ ID NO. 213.

In one embodiment, the present disclosure provides a single chain humanantibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions,wherein the heavy chain variable domain sequence that is at least 95%identical, at least 96% identical, at least 97% identical, at least 98%identical, at least 99% identical, or 100% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO.201, and that has a light chain variable domain sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, at least 99%, or 100% identical to the amino acidsequence consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ IDNO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23,SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO.30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ IDNO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209and SEQ ID NO. 213. Preferably, the human single chain antibody has botha heavy chain variable domain region and a light chain variable domainregion, wherein the single chain human antibody has a heavy chain/lightchain variable domain sequence selected from the group consisting of SEQID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ IDNO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, SEQ ID NO. 7/SEQ ID NO. 17, SEQ ID NO. 3/SEQ ID NO. 18, SEQ ID NO.19/SEQ ID NO. 20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 7/SEQ ID NO.23, SEQ ID NO. 24/SEQ ID NO. 25, SEQ ID NO. 7/SEQ ID NO. 26, SEQ ID NO.7/SEQ ID NO. 27, SEQ ID NO. 7/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO.30, SEQ ID NO. 31/SEQ ID NO. 32, SEQ ID NO. 3/SEQ ID NO. 33, SEQ ID NO.34/SEQ ID NO. 35, SEQ ID NO. 7/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO.38, SEQ ID NO. 39/SEQ ID NO. 40, SEQ ID NO. 7/SEQ ID NO. 41, SEQ ID NO.42/SEQ ID NO. 43), SEQ ID NO. 44/SEQ ID NO. 45, SEQ ID NO. 46/SEQ ID NO.47 and SEQ ID NO. 7/SEQ ID NO. 48, SEQ ID NO. 193/SEQ ID NO. 194, SEQ IDNO. 201/SEQ ID NO. 202, SEQ ID NO. 5/SEQ ID NO. 209 and SEQ ID NO. 7/SEQID NO. 213.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP→CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies. Thus, inone embodiment, the antibody of the invention is a human IgG1 antibody.Thus, in one embodiment, the antibody of the invention is a human IgG4antibody.

The present disclosure provides a number of antibodies structurallycharacterized by the amino acid sequences of their variable domainregions. However, the amino acid sequences can undergo some changeswhile retaining their high degree of binding to their specific targets.More specifically, many amino acids in the variable domain region can bechanged with conservative substitutions and it is predictable that thebinding characteristics of the resulting antibody will not differ fromthe binding characteristics of the wild type antibody sequence. Thereare many amino acids in an antibody variable domain that do not directlyinteract with the antigen or impact antigen binding and are not criticalfor determining antibody structure. For example, a predictednonessential amino acid residue in any of the disclosed antibodies ispreferably replaced with another amino acid residue from the same class.Methods of identifying amino acid conservative substitutions which donot eliminate antigen binding are well-known in the art (see, e.g.,Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al. ProteinEng. 12(10):879-884 (1999); and Burks et al. Proc. Natl. Acad. Sci. USA94:412-417 (1997)). Near et al. Mol. Immunol. 30:369-377, 1993 explainshow to impact or not impact binding through site-directed mutagenesis.Near et al. only mutated residues that they thought had a highprobability of changing antigen binding. Most had a modest or negativeeffect on binding affinity (Near et al. Table 3) and binding todifferent forms of digoxin (Near et al. Table 2).

In certain embodiments, an antibody, or antigen-binding fragmentthereof, of the invention has a dissociation constant (K_(D)) of 1×10⁻⁶M or less; 5×10⁻⁷ M or less; 1×10⁻⁷ M or less; 5×10⁻⁸ M or less; 1×10⁻⁸M or less; 5×10⁻⁹ M or less; or 1×10⁻⁹ M or less. In one embodiment, theantibody, or antigen-binding fragment thereof, of the invention as aK_(D) from 1×10⁻⁷ M to 1×10⁻¹⁰ M. In one embodiment, the antibody, orantigen-binding fragment thereof, of the invention as a K_(D) from1×10⁻⁸ M to 1×10⁻¹⁰ M.

Those of ordinary skill in the art will appreciate standard methodsknown for determining the K_(D) of an antibody, or fragment thereof. Forexample, in one embodiment, K_(D) is measured by a radiolabeled antigenbinding assay (RIA). In one embodiment, an RIA is performed with the Fabversion of an antibody of interest and its antigen. For example,solution binding affinity of Fabs for antigen is measured byequilibrating Fab with a minimal concentration of (¹²⁵I)-labeled antigenin the presence of a titration series of unlabeled antigen, thencapturing bound antigen with an anti-Fab antibody-coated plate (see,e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)).

According to another embodiment, K_(D) is measured using a BIACOREsurface plasmon resonance assay. The term “surface plasmon resonance”,as used herein, refers to an optical phenomenon that allows for theanalysis of real-time interactions by detection of alterations inprotein concentrations within a biosensor matrix, for example using theBIACORE system (Biacore Life Sciences division of GE Healthcare,Piscataway, N.J.). Surface plasmon resonance can also be used todetermine K_(off) and K_(a) values.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for OX40 of at least 10³ M⁻¹S⁻¹. In other embodiments, the antigen binding proteins exhibit a K_(a)of at least 10³ M⁻¹ S⁻¹, at least 10⁴ M⁻¹ S⁻¹, at least 10⁵ M⁻¹ S⁻¹, orat least 10⁶ M⁻¹ S⁻¹. In other further embodiments, the antigen bindingproteins exhibit a K_(a) of at least 10⁷ M⁻¹ S⁻¹. In other furtherembodiments, the antigen binding proteins exhibit a K_(a) of at least10⁷ M⁻¹ S⁻¹ or at least 10⁸ M⁻¹ S⁻¹. In one embodiment, the anti-OX40antibody, or fragment thereof, of the invention has a K_(a) of at least10³-10⁷ M⁻¹ S⁻¹. In another embodiment, the antigen binding proteinexhibits a K_(a) substantially the same as that of an antibody describedherein in the Examples. K_(a) can be determined by Biacore testing, forexample with Biacore 3000 or T200.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from OX40. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to 10⁻¹sec⁻¹ or lower. In another embodiment, the K_(off) is 5×10⁻⁵ to 10⁻¹sec⁻¹ or lower. In another embodiment, the K_(off) is 5×10⁻⁶ to 10⁻¹sec⁻¹ or lower. In another embodiment, the K_(off) is substantially thesame as an antibody described herein. In another embodiment, the antigenbinding protein binds to OX40 with substantially the same K_(off) as anantibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of OX40. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower; in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of OX40with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to OX40 expressed on the surface of a cell and, whenso bound, inhibits OX40 signaling activity in the cell without causing asignificant reduction in the amount of OX40 on the surface of the cell.Any method for determining or estimating the amount of OX40 on thesurface and/or in the interior of the cell can be used. In otherembodiments, binding of the antigen binding protein to theOX40-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface OX40 to be internalized.In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of OX40, or to anepitope of OX40 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise a OX40 binding site from one ofthe herein-described antibodies and a second OX40 binding region fromanother of the herein-described antibodies, including those describedherein by reference to other publications. Alternatively, a bispecificantigen binding protein may comprise an antigen binding site from one ofthe herein described antibodies and a second antigen binding site fromanother OX40 antibody that is known in the art, or from an antibody thatis prepared by known methods or the methods described herein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold) bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyurrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Oligomers that contain one or more antigen binding proteins may beemployed as OX40 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have OX40 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-OX40 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-OX40 antibody fragments or derivatives that formare recovered from the culture supernatant.

Antigen binding proteins directed against OX40 can be used, for example,in assays to detect the presence of OX40 polypeptides, either in vitroor in vivo. The antigen binding proteins also may be employed inpurifying OX40 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as OX40 antagonists may beemployed in treating any OX40-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit OX40-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic of OX40, examples of which are provided herein, thus may betreated. In one embodiment, the present invention provides a therapeuticmethod comprising in vivo administration of a OX40 blocking antigenbinding protein to a mammal in need thereof in an amount effective forreducing a OX40-induced biological activity.

In certain embodiments of the invention, antigen binding proteinsinclude human monoclonal antibodies that inhibit a biological activityof OX40.

Antigen binding proteins, including antibodies and antibody fragmentsdescribed herein, may be prepared by any of a number of conventionaltechniques. For example, they may be purified from cells that naturallyexpress them (e.g., an antibody can be purified from a hybridoma thatproduces it), or produced in recombinant expression systems, using anytechnique known in the art. See, for example, Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980); and Antibodies: A LaboratoryManual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides, including antibodies and antibody fragmentsdescribed herein, of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of OX40bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-OX40 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-OX40 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Antibodies and fragments thereof of the present disclosure can beproduced using any standard methods known in the art. In one example,the polypeptides are produced by recombinant DNA methods by inserting anucleic acid sequence (a cDNA) encoding the polypeptide into arecombinant expression vector and expressing the DNA sequence underconditions promoting expression. The invention includes nucleic acidsencoding any of the polypeptide sequences described in SEQ ID Nos: 1 to216, as well as vectors comprising said nucleic acid sequences.

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants is additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins can also be produced using cell-translation systems. For suchpurposes the nucleic acids encoding the polypeptide must be modified toallow in vitro transcription to produce mRNA and to allow cell-freetranslation of the mRNA in the particular cell-free system beingutilized (eukaryotic such as a mammalian or yeast cell-free translationsystem or prokaryotic such as a bacterial cell-free translation system.

OX40-binding polypeptides can also be produced by chemical synthesis(such as by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

In certain embodiments, the present disclosure provides monoclonalantibodies that bind to OX40. Monoclonal antibodies may be producedusing any technique known in the art, e.g., by immortalizing spleencells harvested from the transgenic animal after completion of theimmunization schedule. The spleen cells can be immortalized using anytechnique known in the art, e.g., by fusing them with myeloma cells toproduce hybridomas. Myeloma cells for use in hybridoma-producing fusionprocedures preferably are non-antibody-producing, have high fusionefficiency, and enzyme deficiencies that render them incapable ofgrowing in certain selective media which support the growth of only thedesired fused cells (hybridomas). Examples of suitable cell lines foruse in mouse fusions include Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag4 1, Sp210-Ag14, FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XXO Bul;examples of cell lines used in rat fusions include R210.RCY3, Y3-Ag1.2.3, IR983F and 48210. Other cell lines useful for cell fusions areU-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

In one embodiment, modified forms of the subject soluble polypeptidescomprise linking the subject soluble polypeptides to nonproteinaceouspolymers. In one embodiment, the polymer is polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner as set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)—CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf-life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Methods, Formulations and Modes of Administration

Any of the anti-OX40 antibodies or antigen binding fragments disclosedherein may be used in such therapeutic methods. Examples of anti-OX40antibodies and antigen binding fragments that may be used in thetherapeutic methods and compositions of the invention are describedabove.

In one embodiment, the anti-OX40 antibodies and antibody fragments ofthe invention are used to treat a disease requiring either stimulationof immune responses or suppression. OX40 stimulation (with agonists) isuseful in treating a disease where the immune response (preferentially Tcells) needs to be boosted. For example, in treating cancer. On thecontrary OX40/OX40L blockade is useful in treating a disease in whichinflammation needs to be reduced, for example during autoimmunity orallergy. OX40/OX40L blockade is usually done by using OX40L blockingantibodies.

In certain embodiments, the disease is selected from the groupconsisting of cancers, autoimmune diseases and infections.

In certain embodiments, anti-OX40 antibodies and antibody fragments ofthe invention may be used to treat cancer.

In other embodiments, anti-OX40 antibodies and antibody fragments of theinvention may be used in treating infection (e.g., infection with abacteria or virus or other pathogen). In some embodiments, the infectionis with a virus and/or a bacteria. In some embodiments, the infection iswith a pathogen.

In other embodiments, anti-OX40 antibodies and antibody fragments of theinvention may be used to enhance immune function (e.g., by upregulatingcell-mediated immune responses), e.g., in an individual having cancer.

The present disclosure further provides a method for treating a broadspectrum of mammalian cancers, infectious diseases, or autoimmunereactions, comprising administering an anti-OX40 polypeptide using theantibodies, and antibody fragments, disclosed herein. In one embodiment,the invention provides a method of treating cancer by administering ananti-human OX40 antibody to a subject in need thereof. Examples ofantibodies, and fragments thereof, that may be used in the therapeuticsmethods disclosed herein include an anti-human OX40 human antibody of anIgG class having a binding affinity of at least 10⁻⁶M, or an anti-humanOX40 Fab antibody fragment comprising a heavy chain variable region anda light chain variable region from the antibody sequences described inSEQ ID Nos. 1-48, 193, 194, 201, 202, 209 and 213 or comprising the CDRsdescribed in any of the antibody sequences of SEQ ID Nos: 1-48, 193,194, 201, 202, 209 and 213. In one embodiment, the methods disclosedherein comprise administering an isolated human antibody comprising aheavy chain variable domain sequence that is at least 95% identical, atleast 96% identical, at least 97% identical, at least 98% identical, orat least 99% identical, to an amino acid sequence selected from thegroup consisting of heavy chain and a variable domain region from alight chain, wherein the heavy chain variable domain sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, at least 99%, or 100% identical, to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO.201, and having a light chain variable domain sequence that is at least95% identical, at least 96% identical, at least 97% identical, at least98% identical, or at least 99% identical, to an amino acid sequenceselected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ IDNO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ IDNO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28,SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO.36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ IDNO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202,SEQ ID NO. 209 and SEQ ID NO. 213. In one embodiment, the methodsdisclosed herein comprise administering an IgG human anti-hOX40 antibodycomprising a heavy chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO.7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, and having a light chainvariable domain sequence selected form the group consisting of SEQ IDNO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26,SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO.33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ IDNO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In one embodiment, the methods described herein include the use of a Fabfragment comprising a heavy chain variable domain sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical, to an amino acidsequence selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 5, SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO.13, SEQ ID NO. 15, SEQ ID NO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ IDNO. 29, SEQ ID NO. 31, SEQ ID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQID NO. 42, SEQ ID NO. 44, SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO.201, and comprising a light chain variable domain sequence that is atleast 95% identical, at least 96% identical, at least 97% identical, atleast 98% identical, or at least 99% identical, to an amino acidsequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO.4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO.14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ IDNO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35,SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO.43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ IDNO. 202, SEQ ID NO. 209 and SEQ ID NO. 213. In one embodiment, themethods described herein include the use of a human Fab antibodyfragment comprising a heavy chain variable domain sequence selected fromthe group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ IDNO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ IDNO. 19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQID NO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44,SEQ ID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, and comprising a lightchain variable domain sequence selected from the group consisting of SEQID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18,SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 25, SEQ ID NO.26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQ ID NO. 32, SEQ IDNO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38, SEQ ID NO. 40, SEQID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO. 47, SEQ ID NO. 48,SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 and SEQ ID NO. 213.

In one embodiment, the methods described herein include the use of asingle chain human antibody, e.g., scFv, comprising a heavy chainvariable domain sequence that is at least 95% identical, at least 96%identical, at least 97% identical, at least 98% identical, or at least99% identical, to an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, and comprising a lightchain variable domain sequence that is at least 95% identical, at least96% identical, at least 97% identical, at least 98% identical, or atleast 99% identical, to an amino acid sequence selected from the groupconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ ID NO.17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQ IDNO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30, SEQID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 38,SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ ID NO.47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209 andSEQ ID NO. 213. In one embodiment, the methods described herein includethe use of a single chain human antibody comprising a heavy chainvariable domain having an amino acid sequence selected from the groupconsisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 5, SEQ ID NO. 7,SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, SEQ ID NO.19, SEQ ID NO. 21, SEQ ID NO. 24, SEQ ID NO. 29, SEQ ID NO. 31, SEQ IDNO. 34, SEQ ID NO. 37, SEQ ID NO. 39, SEQ ID NO. 42, SEQ ID NO. 44, SEQID NO. 46, SEQ ID NO. 193 and SEQ ID NO. 201, and comprising a lightchain variable domain having an amino acid sequence selected from thegroup consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO.8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, SEQ IDNO. 17, SEQ ID NO. 18, SEQ ID NO. 20, SEQ ID NO. 22, SEQ ID NO. 23, SEQID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 30,SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO.38, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 43, SEQ ID NO. 45, SEQ IDNO. 47, SEQ ID NO. 48, SEQ ID NO. 194, SEQ ID NO. 202, SEQ ID NO. 209and SEQ ID NO. 213.

In one embodiment, the isolated anti-OX40 human antibody used in themethods of the invention comprises a heavy chain and a light chainwherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called OX1A11 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called OX1B1 herein),SEQ ID NO. 5/SEQ ID NO. 6 (called OX1B2 herein), SEQ ID NO. 7/SEQ ID NO.8 (called OX1B3 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called OX1C4herein), SEQ ID NO. 11/SEQ ID NO. 12 (called OX1C5 herein), SEQ ID NO.13/SEQ ID NO. 14 (called OX1D9 herein), SEQ ID NO. 15/SEQ ID NO. 16(called OX1E10 herein), SEQ ID NO. 7/SEQ ID NO. 17 (called OX1E7herein), SEQ ID NO. 3/SEQ ID NO. 18 (called OX1F2 herein), SEQ ID NO.19/SEQ ID NO. 20 (called OX1G9), SEQ ID NO. 21/SEQ ID NO. 22 (calledOX2B12 herein), SEQ ID NO. 7/SEQ ID NO. 23 (called OX2B3 herein), SEQ IDNO. 24/SEQ ID NO. 25 (called OX2B4 herein), SEQ ID NO. 7/SEQ ID NO. 26(called OX2B6 herein), SEQ ID NO. 7/SEQ ID NO. 27 (called OX2F2 herein),SEQ ID NO. 7/SEQ ID NO. 28 (called OX2G2 herein), SEQ ID NO. 29/SEQ IDNO. 30 (called OX3C10 herein), SEQ ID NO. 31/SEQ ID NO. 32 (calledOX4A11 herein), SEQ ID NO. 3/SEQ ID NO. 33 (called OX4Al2 herein), SEQID NO. 34/SEQ ID NO. 35 (called OX4B6 herein), SEQ ID NO. 7/SEQ ID NO.36 (called OX4D4 herein), SEQ ID NO. 37/SEQ ID NO. 38 (called OX4D7herein), SEQ ID NO. 39/SEQ ID NO. 40 (called OX4D9 herein), SEQ ID NO.7/SEQ ID NO. 41 (called OX4G9 herein), SEQ ID NO. 42/SEQ ID NO. 43(called OX4H4 herein), SEQ ID NO. 44/SEQ ID NO. 45 (called OX5B9herein), SEQ ID NO. 46/SEQ ID NO. 47 (called OX5C1 herein), SEQ ID NO.7/SEQ ID NO. 48 (called OX5D7 herein), SEQ ID NO. 193/SEQ ID NO. 194(called OX4B5 herein), SEQ ID NO. 201/SEQ ID NO. 202 (called OX2E5herein), SEQ ID NO. 5/SEQ ID NO. 209 (called OX2B5 herein) and SEQ IDNO.7/SEQ ID NO. 213 (called OX5C11 herein).

In one embodiment, a Fab fragment used in the methods of the inventionhas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 (called OX1A11 herein), SEQ ID NO. 3/SEQ ID NO. 4(called OX1B1 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called OX1B2 herein),SEQ ID NO. 7/SEQ ID NO. 8 (called OX1B3 herein), SEQ ID NO. 9/SEQ ID NO.10 (called OX1C4 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called OX1C5herein), SEQ ID NO. 13/SEQ ID NO. 14 (called OX1D9 herein), SEQ ID NO.15/SEQ ID NO. 16 (called OX1E10 herein), SEQ ID NO. 7/SEQ ID NO. 17(called OX1E7 herein), SEQ ID NO. 3/SEQ ID NO. 18 (called OX1F2 herein),SEQ ID NO. 19/SEQ ID NO. 20 (called OX1G9), SEQ ID NO. 21/SEQ ID NO. 22(called OX2B12 herein), SEQ ID NO. 7/SEQ ID NO. 23 (called OX2B3herein), SEQ ID NO. 24/SEQ ID NO. 25 (called OX2B4 herein), SEQ ID NO.7/SEQ ID NO. 26 (called OX2B6 herein), SEQ ID NO. 7/SEQ ID NO. 27(called OX2F2 herein), SEQ ID NO. 7/SEQ ID NO. 28 (called OX2G2 herein),SEQ ID NO. 29/SEQ ID NO. 30 (called OX3C10 herein), SEQ ID NO. 31/SEQ IDNO. 32 (called OX4A11 herein), SEQ ID NO. 3/SEQ ID NO. 33 (called OX4Al2herein), SEQ ID NO. 34/SEQ ID NO. 35 (called OX4B6 herein), SEQ ID NO.7/SEQ ID NO. 36 (called OX4D4 herein), SEQ ID NO. 37/SEQ ID NO. 38(called OX4D7 herein), SEQ ID NO. 39/SEQ ID NO. 40 (called OX4D9herein), SEQ ID NO. 7/SEQ ID NO. 41 (called OX4G9 herein), SEQ ID NO.42/SEQ ID NO. 43 (called OX4H4 herein), SEQ ID NO. 44/SEQ ID NO. 45(called OX5B9 herein), SEQ ID NO. 46/SEQ ID NO. 47 (called OX5C1herein), SEQ ID NO. 7/SEQ ID NO. 48 (called OX5D7 herein), SEQ ID NO.193/SEQ ID NO. 194 (called OX4B5 herein), SEQ ID NO. 201/SEQ ID NO. 202(called OX2E5 herein), SEQ ID NO. 5/SEQ ID NO. 209 (called OX2B5herein), and SEQ ID NO. 7/SEQ ID NO. 213 (called OX5C11 herein).

In one embodiment, a human single chain antibody has both a heavy chainvariable domain region and a light chain variable domain region, whereinthe single chain human antibody has a heavy chain/light chain variabledomain sequence selected from the group consisting of SEQ ID NO. 1/SEQID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ IDNO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO.12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, SEQ ID NO.7/SEQ ID NO. 17, SEQ ID NO. 3/SEQ ID NO. 18, SEQ ID NO. 19/SEQ ID NO.20, SEQ ID NO. 21/SEQ ID NO. 22, SEQ ID NO. 7/SEQ ID NO. 23, SEQ ID NO.24/SEQ ID NO. 25, SEQ ID NO. 7/SEQ ID NO. 26, SEQ ID NO. 7/SEQ ID NO.27, SEQ ID NO. 7/SEQ ID NO. 28, SEQ ID NO. 29/SEQ ID NO. 30, SEQ ID NO.31/SEQ ID NO. 32, SEQ ID NO. 3/SEQ ID NO. 33, SEQ ID NO. 34/SEQ ID NO.35, SEQ ID NO. 7/SEQ ID NO. 36, SEQ ID NO. 37/SEQ ID NO. 38, SEQ ID NO.39/SEQ ID NO. 40, SEQ ID NO. 7/SEQ ID NO. 41, SEQ ID NO. 42/SEQ ID NO.43), SEQ ID NO. 44/SEQ ID NO. 45, SEQ ID NO. 46/SEQ ID NO. 47 and SEQ IDNO. 7/SEQ ID NO. 48, SEQ ID NO. 193/SEQ ID NO. 194, SEQ ID NO. 201/SEQID NO. 202, SEQ ID NO. 5/SEQ ID NO. 209 and SEQ ID NO. 7/SEQ ID NO. 213.

Cancer Indications

Anti-OX40 antibodies and antibody fragments of the invention may be usedto treat a subject having cancer, including, for example, a solid tumoror leukemic cell that expresses OX40 on the cell surface.

In one embodiment, the OX40 antibodies and antibody fragments describedherein are useful in treating, delaying the progression of, preventingrelapse of or alleviating a symptom of a cancer or other neoplasticcondition in a subject in need thereof.

In one embodiment, a cancer that may be treated using the antibodies andfragments of the invention, include, but are not limited to, prostatecancer, breast cancer, ovarian cancer, head and neck cancer, bladdercancer, melanoma, colorectal cancer, pancreatic cancer, lung cancer,leiomyoma, leiomyo sarcoma, glioma, glioblastoma, esophageal cancer,liver cancer, kidney cancer, stomach cancer, colon cancer, cervicalcancer, uterine cancer, liver cancer and a hematological cancer.

As used herein, “hematological cancer” refers to a cancer of the blood,and includes leukemia, lymphoma and myeloma among others. “Leukemia”refers to a cancer of the blood in which too many white blood cells thatare ineffective in fighting infection are made, thus crowding out theother parts that make up the blood, such as platelets and red bloodcells. It is understood that cases of leukemia are classified as acuteor chronic. “Lymphoma” refers to a group of blood cancers that developin the lymphatic system. “Myeloma” refers to a cancer that forms in atype of white blood cell called a plasma cell.

Certain forms of leukemia that may be treated using the antibodies andfragments of the invention, include B chronic lymphocytic leukemia(B-CLL), B and T acute lymphocytic leukemia (ALL); acute myeloidleukemia (AML); chronic lymphocytic leukemia (CLL); chronic myelogenousleukemia (CML); hairy cell leukemia (HCL); Myeloproliferativedisorder/neoplasm (MPDS); and myelodysplasia syndrome.

Certain forms of lymphoma that may be treated using the antibodies andfragments of the invention include B-cell lymphomas, including lowgrade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL)NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL;high grade immunoblastic NHL; high grade lymphoblastic NHL; high gradesmall non-cleaved cell NHL; bulky disease NHL; relapsed aggressive NHL,relapsed indolent NHL, refractory NHL, refractory indolent NHL,Burkitt's lymphoma, mantle cell lymphoma, AIDS-related lymphoma; andWaldenstrom's Macroglobulinemia, T cell lymphoma (TCL) and Hodgkin'sLymphoma (HL) among others.

Certain forms of myeloma that may be treated using the antibodies andfragments of the invention include multiple myeloma (MM), plasma cellmyeloma, plamocytoma, giant cell myeloma, heavy-chain myeloma, and lightchain or Bence-Jones myeloma.

Blockade of OX40 by antibodies can enhance an immune response againstcancerous cells in the patient. An anti-OX40 antibody or antibodyfragment disclosed herein can be used alone to inhibit the growth ofcancerous tumors.

Alternatively, an anti-OX40 antibody or antibody fragment disclosedherein can be used in conjunction with other immunogenic agents,standard cancer treatments, or other antibodies. In one embodiment, thepresent disclosure provides a method of inhibiting growth of tumor cellsin a subject, comprising administering to the subject a therapeuticallyeffective amount of an anti-OX40 antibody, or antigen-binding fragmentthereof. Preferably, the antibody or antibody fragment is a humananti-OX40 antibody or antibody fragment (such as any of the humananti-OX40 antibodies described herein).

In one embodiment, preferred cancers whose growth may be inhibitedinclude cancers typically responsive to immunotherapy.

Optionally antibodies and antibody fragments to OX40 described hereincan be combined with an immunogenic agent, such as cancerous cells,purified tumor antigens (including recombinant proteins, peptides, andcarbohydrate molecules), cells, and cells transfected with genesencoding immune stimulating cytokines (He et al (2004) J. Immunol.173:4919-28). Non-limiting examples of tumor vaccines that can be usedinclude peptides of melanoma antigens, such as peptides of gp100, MAGEantigens, Trp-2, MART1 and/or tyrosinase, or tumor cells transfected toexpress the cytokine GM-CSF (discussed further below).

In humans, some tumors have been shown to be immunogenic such asmelanomas.

In certain embodiments, an anti-OX40 antibody or antibody fragmentdisclosed herein may be combined with a vaccination protocol. Manyexperimental strategies for vaccination against tumors have beendevised. In one of these strategies, a vaccine is prepared usingautologous or allogeneic tumor cells. These cellular vaccines have beenshown to be most effective when the tumor cells are transduced toexpress GM-CSF. GM-CSF has been shown to be a potent activator ofantigen presentation for tumor vaccination (Dranoff et al. (1993) Proc.Natl. Acad. Sci U.S.A. 90: 3539-43).

The study of gene expression and large scale gene expression patterns invarious tumors has led to the definition of so called tumor specificantigens (Rosenberg, S A (1999) Immunity 10: 281-7). In many cases,these tumor specific antigens are differentiation antigens expressed inthe tumors and in the cell from which the tumor arose, for examplemelanocyte antigens gp100, MAGE antigens, and Trp-2. Many of theseantigens can be shown to be the targets of tumor specific T cells foundin the host. An anti-OX40 antibody or antibody fragment disclosed hereincan be used in conjunction with a collection of recombinant proteinsand/or peptides expressed in a tumor in order to generate an immuneresponse to these proteins. These proteins are normally viewed by theimmune system as self antigens and are therefore tolerant to them. Thetumor antigen can include the protein telomerase, which is required forthe synthesis of telomeres of chromosomes and which is expressed in morethan 85% of human cancers and in only a limited number of somatictissues (Kim et al. (1994) Science 266: 2011-2013). (These somatictissues may be protected from immune attack by various means). Tumorantigen can also be “neo-antigens” expressed in cancer cells because ofsomatic mutations that alter protein sequence or create fusion proteinsbetween two unrelated sequences (i.e., bcr-abl in the Philadelphiachromosome), or idiotype from B cell tumors.

Other tumor vaccines can include the proteins from viruses implicated inhuman cancers such a Human Papilloma Viruses (HPV), Hepatitis Viruses(HBV and HCV) and Kaposi's Herpes Sarcoma Virus (KHSV). Another form oftumor specific antigen which can be used in conjunction with ananti-OX40 antibody or antibody fragment disclosed herein is purifiedheat shock proteins (HSP) isolated from the tumor tissue itself. Theseheat shock proteins contain fragments of proteins from the tumor cellsand these HSPs are highly efficient at delivery to antigen presentingcells for eliciting tumor immunity (Suot & Srivastava (1995) Science269:1585-1588; Tamura et al. (1997) Science 278:117-120).

Dendritic cells (DC) are potent antigen presenting cells that can beused to prime antigen-specific responses. DC's can be produced ex vivoand loaded with various protein and peptide antigens as well as tumorcell extracts (Nestle et al. (1998) Nature Medicine 4: 328-332). DCs canalso be transduced by genetic means to express these tumor antigens aswell. DCs have also been fused directly to tumor cells for the purposesof immunization (Kugler et al. (2000) Nature Medicine 6:332-336). As amethod of vaccination, DC immunization can be effectively combined withOX40 blockade to activate more potent anti-tumor responses.

An anti-OX40 antibody or antibody fragment disclosed herein can also becombined with other cancer treatments. An anti-OX40 antibody or antibodyfragment disclosed herein can be effectively combined withchemotherapeutic regimes. In these instances, it may be possible toreduce the dose of chemotherapeutic reagent administered (Mokyr et al.(1998) Cancer Research 58: 5301-5304). An example of such a combinationis an anti-OX40 antibody in combination with decarbazine for thetreatment of melanoma. Another example of such a combination is ananti-OX40 antibody in combination with interleukin-2 (IL-2) for thetreatment of melanoma. The scientific rationale behind the combined useof OX40 blockade and chemotherapy is that cell death, that is aconsequence of the cytotoxic action of most chemotherapeutic compounds,should result in increased levels of tumor antigen in the antigenpresentation pathway. Other combination therapies that may result insynergy with an anti-OX40 antibody or antibody fragment disclosed hereinthrough cell death are radiation, surgery, and hormone deprivation. Eachof these protocols creates a source of tumor antigen in the host.Angiogenesis inhibitors can also be combined with an anti-OX40 antibodyor antibody fragment disclosed herein. Inhibition of angiogenesis oftenleads to tumor cell death which may feed tumor antigens into hostantigen presentation pathways.

Bispecific antibodies can be used to target two separate antigens. Forexample anti-Fc receptor/anti-tumor antigen (e.g., Her-2/neu) bispecificantibodies have been used to target macrophages to sites of tumor. Thistargeting may more effectively activate tumor specific responses. The Tcell arm of these responses would be augmented by the use of ananti-OX40 antibody or antibody fragment disclosed herein. Alternatively,antigen may be delivered directly to DCs by the use of bispecificantibodies which bind to tumor antigen and a dendritic cell specificcell surface marker.

Bispecific antibodies can be used to target two separate tumor antigens.A variety of tumor targets may be considered, including, for example,Her2, cMet, EGFR and VEGFR expressing tumors. As such, in oneembodiment, the invention provides a bispecific antibody comprising ananti-OX40 antibody (or antigen binding fragment) comprising a heavy andlight chain variable region sequence as described herein or a heavy andlight chain variable region comprising a set of CDR sequences asdescribed herein and an anti-Her2, an anti-EGFR, an anti-VEGFR (see, forexamples, antibodies described in U.S. Pat. No. 9,029,510, incorporatedby reference herein), or an anti-cMet antibody (or antigen bindingportion thereof). In one embodiment, the invention includes a bispecificantibody specific to OX40 and EGFR, wherein the antibody comprises ananti-OX40 antibody or fragment as disclosed herein and an anti-EGFRantibody or fragment as described in International Publication No. WO2013/173255 or International Publication No. WO 2014/066530, both ofwhich are incorporated by reference in their entireties herein. In oneembodiment, the invention includes a bispecific antibody specific toOX40 and VEGFR, wherein the antibody comprises an anti-OX40 antibody orfragment as disclosed herein and an anti-VEGFR antibody or fragment asdescribed in U.S. Pat. No. 9,029,510, incorporated by reference in itsentirety herein. In one embodiment, the invention includes a bispecificantibody specific to OX40 and cMet, wherein the antibody comprises ananti-OX40 antibody or fragment as disclosed herein and an anti-cMetantibody or fragment as described in International Publication No. WO2016/094455, incorporated by reference in its entirety herein.

Anti-OX40 antibodies or antibody fragments disclosed herein can also beused in combination with bispecific antibodies that target, for example,Fcα or Fcγ receptor-expressing effectors cells to tumor cells (U.S. Pat.Nos. 5,922,845 and 5,837,243).

Tumors evade host immune surveillance by a large variety of mechanisms.Many of these mechanisms may be overcome by the inactivation of proteinswhich are expressed by the tumors and which are immunosuppressive. Theseinclude among others TGF-β (Kehrl et al. (1986) J. Exp. Med. 163:1037-1050), IL-10 (Howard & O'Garra (1992) Immunology Today 13:198-200), and Fas ligand (Hahne et al. (1996) Science 274: 1363-1365).Antibodies to each of these entities can be used in combination withanti-OX40 antibodies and antibody fragments described herein tocounteract the effects of the immunosuppressive agent and favor tumorimmune responses by the host.

Other antibodies which activate host immune responsiveness can be usedin combination with anti-OX40 antibodies and antibody fragmentsdescribed herein. These include molecules on the surface of dendriticcells which activate DC function and antigen presentation. Anti-CD40antibodies are able to substitute effectively for T cell helper activity(Ridge et al. (1998) Nature 393: 474-478) and can be used in conjunctionwith OX40 antibodies (Ito et al. (2000) Immunobiology 201 (5) 527-40).Activating antibodies to T cell costimulatory molecules such as CTLA-4,OX-40, 4-1BB, and ICOS may also provide for increased levels of T cellactivation. OX40 blockade can be used to increase the effectiveness ofthe donor engrafted tumor specific T cells.

There are also several experimental treatment protocols that involve exvivo activation and expansion of antigen specific T cells and adoptivetransfer of these cells into recipients in order to stimulateantigen-specific T cells against tumor (Greenberg & Riddell (1999)Science 285: 546-51). These methods can also be used to activate T cellresponses to infectious agents such as CMV. Ex vivo activation in thepresence of anti-OX40 antibodies can increase the frequency and activityof the adoptively transferred T cells.

Additional methods for treating cancer using the anti-OX40 antibodiesand fragments of the invention are disclosed below, for example, in theCombination Therapy section.

Infections

The present disclosure further provides a method of treating aninfectious disease in a subject comprising administering to the subjectan anti-OX40 antibody, or antigen-binding portion thereof, such that thesubject is treated for the infectious disease. Preferably, the antibodyis a human anti-human OX40 antibody or antibody fragment (such as any ofthe human anti-OX40 antibodies described herein).

In other embodiments, anti-OX40 antibodies and antibody fragments of theinvention may be used in treating infection (e.g., infection with abacteria or virus or other pathogen). In some embodiments, the infectionis with a virus and/or a bacteria. In some embodiments, the infection iswith a pathogen.

Some examples of pathogenic viruses causing infections treatable by thedisclosed antibodies include HIV, hepatitis (A, B, or C), herpes virus(e.g., VZV, HSV-1, HAV-6, HSV-II, and CMV, Epstein Barr virus),adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, molluscum virus, poliovirus,rabies virus, JC virus and arboviral encephalitis virus.

Some examples of pathogenic bacteria causing infections treatable by thedisclosed antibodies include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, streptococci, pneumonococci, meningococciand gonococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria.

Combination Therapy

In one embodiment, the anti-OX40 antibodies and antibody fragments ofthe invention can be administered alone or in combination with one ormore additional therapies. Long-term therapy is equally possible as isadjuvant therapy in the context of other treatment strategies, asdescribed above.

In some embodiments, the anti-OX40 antibodies and antibody fragments ofthe invention may be administered in conjunction with a chemotherapy orchemotherapeutic agent. In some embodiments, the anti-OX40 antibodiesand antibody fragments of the invention may be administered inconjunction with a radiation therapy or radiotherapeutic agent. In someembodiments, the anti-OX40 antibodies and antibody fragments of theinvention may be administered in conjunction with a targeted therapy ortargeted therapeutic agent. In some embodiments, the anti-OX40antibodies and antibody fragments of the invention may be administeredin conjunction with an immunotherapy or immunotherapeutic agent, forexample a monoclonal antibody.

In certain embodiments of such methods, one or more anti-OX40 antibodiesand antibody fragments of the invention can be administered, together(simultaneously) or at different times (sequentially). In addition,anti-OX40 antibodies and antibody fragments of the invention can beadministered with another type of compound(s) for treating cancer or forinhibiting angiogenesis.

The disclosed human anti-OX40 antibodies can be co-administered with oneor other more therapeutic agents, e.g., a cytotoxic agent, a radiotoxicagent or an immunosuppressive agent. The antibody can be linked to theagent (as an immuno-complex) or can be administered separate from theagent. In the latter case (separate administration), the antibody can beadministered before, after or concurrently with the agent or can beco-administered with other known therapies, e.g., an anti-cancertherapy, e.g., radiation. Such therapeutic agents include, among others,anti-neoplastic agents such as doxorubicin (adriamycin), cisplatinbleomycin sulfate, carmustine, chlorambucil, dacarbazine andcyclophosphamide hydroxyurea which, by themselves, are only effective atlevels which are toxic or subtoxic to a patient. Cisplatin isintravenously administered as a 100 mg/ml dose once every four weeks andadriamycin is intravenously administered as a 60-75 mg/ml dose onceevery 21 days. Co-administration of the anti-OX40 antibodies andantibody fragments of the invention, with chemotherapeutic agentsprovides two anti-cancer agents which operate via different mechanismswhich yield a cytotoxic effect to human tumor cells. Suchco-administration can solve problems due to development of resistance todrugs or a change in the antigenicity of the tumor cells which wouldrender them unreactive with the antibody.

OX40 is not constitutively expressed on naive T cells, but is inducedafter engagement of the T cell receptor (TCR). The ligand for OX40,OX40L, is predominantly expressed on antigen presenting cells. OX40 ishighly expressed by activated CD4+ T cells, activated CD8+ T cells,memory T cells, and regulatory T cells. OX40 signaling can providecostimulatory signals to CD4 and CD8 T cells, leading to enhanced cellproliferation, survival, effector function and migration. OX40 signalingalso enhances memory T cell development and function.

Accordingly, an anti-OX40 antibody or antibody fragment as describedherein, may be co-administered with one or more additional antibodiesthat are effective in stimulating immune responses to thereby furtherenhance, stimulate or upregulate immune responses in a subject. Forexample, the invention provides a method for stimulating an immuneresponse in a subject comprising administering to the subject ananti-OX40 antibody or antibody fragment and one or more additionalimmunostimulatory antibodies, such as an anti-PD-1 antibody, ananti-PD-L1 antibody and/or an anti-CTLA-4 antibody, such that an immuneresponse is stimulated in the subject, for example to inhibit tumorgrowth or to stimulate an anti-viral response.

An important part of the immune system is its ability to distinguishbetween normal cells in the body and those it sees as “foreign.” Thislets the immune system attack the foreign cells while leaving the normalcells alone. To do this, it uses “checkpoints,” which are molecules oncertain immune cells that need to be activated (or inactivated) to startan immune response. Cancer cells sometimes find ways to use thesecheckpoints to avoid being attacked by the immune system. Accordingly,an immune checkpoint inhibitor includes a drug or agent, e.g., anantibody, that can activate T cells which are inactive in the absence ofthe drug or agent due, at least in part, to signaling from a cancer cellwhich can maintain the inactive state of the T cell.

Thus, in one embodiment, an anti-OX40 antibody or antigen bindingantibody fragment of the invention is used in combination with an immunecheckpoint inhibitor for the treatment of cancer. For example, in oneembodiment, an anti-OX40 antibody, or antigen binding fragment,described herein is administered in combination with an antibody whichis an immune checkpoint inhibitor, including, but not limited to, ananti-cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibody, ananti-programmed death 1 (PD-1) antibody, or an anti-programmeddeath-ligand 1 (PD-L1) antibody. In one embodiment, an anti-OX40antibody, or antigen binding fragment, described herein is administeredin combination with trastuzumab (Herceptin).

In one embodiment, the subject is administered an anti-OX40 antibody orantibody fragment and an anti-PD-1 antibody. In one embodiment, thesubject is administered an anti-OX40 antibody or antibody fragmentselected from 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5, and ananti-PD-1 antibody.

In another embodiment, the subject is administered an anti-OX40 antibodyor antibody fragment and an anti-PD-L1 antibody. In one embodiment, thesubject is administered an anti-OX40 antibody or antibody fragmentselected from 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5, and ananti-PD-L1 antibody.

In yet another embodiment, the subject is administered an anti-OX40antibody or antibody fragment and an anti-CTLA-4 antibody. In oneembodiment, the subject is administered an anti-OX40 antibody orantibody fragment selected from 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9,1C4, 4B5, and an anti-CTLA-4 antibody.

In one embodiment, the invention provides a method for treating ahyperproliferative disease (e.g., cancer), comprising administering aOX40 antibody and a CTLA-4 antibody to a subject. In furtherembodiments, the anti-OX40 antibody is administered at a subtherapeuticdose, the anti-CTLA-4 antibody is administered at a subtherapeutic dose,or both are administered at a subtherapeutic dose. Alternatively, amethod for altering an adverse event associated with treatment of ahyperproliferative disease with an immunostimulatory agent, comprisingadministering an anti-OX40 antibody and a subtherapeutic dose ofanti-CTLA-4 antibody to a subject.

In one embodiment, an anti-OX40 antibody, or antigen binding fragment(e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described hereinis administered in combination with an anti-cytotoxic T-lymphocyteantigen 4 (CTLA-4) antibody, for example ipilimumab (YERVOY) ortremelimumab (CP-675,206; MedImmune).

Another combination comprises administering a OX40 antibody or antibodyfragment (e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5) and a PD-1or PD-L1 antibody to a subject. In one embodiment, an anti-OX40antibody, or antigen binding fragment, described herein is administeredin combination with an anti-programmed death 1 (PD-1) antibody, forexample pembrolizumab (KEYTRUDA) or nivolumab (OPDIVO). In oneembodiment, an anti-OX40 antibody, or antigen binding fragment,described herein (e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5) isadministered in combination with an anti-programmed death-ligand 1(PD-L1) antibody, for example avelumab (MSB0010718C), atezolizumab(TECENTRIQ) or durvalumab (MEDI4736). In further embodiments, theanti-OX40 antibody is administered at a subtherapeutic dose, theanti-PD-1 or PD-L1 antibody is administered at a subtherapeutic dose, orboth are administered at a subtherapeutic dose.

In one embodiment, an anti-OX40 antibody, or antigen binding fragment(e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described hereinis administered in conjunction with a PARP inhibitor (e.g., Olaparanib,Rucaparib, Niraparib, Cediranib, BMN673, Veliparib), Trabectedin,nab-paclitaxel (albumen-bound paclitaxel, ABRAXANE), Trebananib,Pazopanib, Cediranib, Palbociclib, everolimus, fluoropyrimidine (e.g.,FOLFOX, FOLFIRI), IFL, regorafenib, Reolysin, Alimta, Zykadia, Sutent,Torisel (temsirolimus), Inlyta (axitinib, Pfizer), Afinitor (everolimus,Novartis), Nexavar (sorafenib, Onyx/Bayer), Votrient, Pazopanib,axitinib, IMA-901, AGS-003, cabozantinib, Vinflunine, Hsp90 inhibitor(e.g., apatorsin), Ad-GM-CSF (CT-0070), Temazolomide, IL-2, IFNa,vinblastine, Thalomid, dacarbazine, cyclophosphamide, lenalidomide,azacytidine, lenalidomide, bortezomid (VELCADE), amrubicine,carfilzomib, pralatrexate, and/or enzastaurin.

In one embodiment, an anti-OX40 antibody, or antigen binding fragment(e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described hereinis administered in conjunction with an agonist directed against anactivating co-stimulatory molecule. In some embodiments, an activatingco-stimulatory molecule may include CD40, CD226, CD28, GITR, CD137,CD27, HVEM, or CD127. In some embodiments, the agonist directed againstan activating co-stimulatory molecule is an agonist antibody that bindsto CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In someembodiments, an anti-human OX40 agonist antibody may be administered inconjunction with an antagonist directed against an inhibitoryco-stimulatory molecule. In some embodiments, an inhibitoryco-stimulatory molecule may include CTLA-4 (also known as CD152), PD-1,TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, orarginase. In some embodiments, the antagonist directed against aninhibitory co-stimulatory molecule is an antagonist antibody that bindsto CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3 (e.g., LAG-3-IgG fusionprotein (IMP321)), B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.

In one embodiment, an anti-OX40 antibody, or antigen binding fragment(e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described hereinis administered in conjunction with an antagonist directed against CD19.In some embodiments, an anti-OX40 antibody, or antigen binding fragment(e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described hereinis administered in conjunction with MOR00208. In some embodiments, ananti-OX40 antibody, or antigen binding fragment (e.g. 1A11, 2E5, 2B12,1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described herein is administered inconjunction with an antagonist directed against CD38.

In one embodiment, an anti-OX40 antibody, or antigen binding fragment(e.g. 1A11, 2E5, 2B12, 1B1, 5C1, 4D7, 5B9, 1C4, 4B5), described hereinis administered in conjunction with an angiogenesis inhibitor (e.g.bevacizumab, sorafenib, sunitinib, pazopanib) and everolimus.

Cancers whose growth may be inhibited using the antibodies of theinstant disclosure include cancers typically responsive toimmunotherapy. Representative examples of cancers for treatment with thecombination therapy of the instant disclosure include those cancersspecifically listed above in the discussion of monotherapy withanti-OX40 antibodies.

Therapeutic Methods and Compositions

Suitable routes of administering the antibody compositions describedherein (e.g., human monoclonal antibodies, multispecific and bispecificmolecules and immunoconjugates) are in vivo and in vitro are well knownin the art and can be selected by those of ordinary skill. For example,the antibody compositions can be administered by injection (e.g.,intravenous or subcutaneous). Suitable dosages of the molecules usedwill depend on the age and weight of the subject and the concentrationand/or formulation of the antibody composition.

Techniques and dosages for administration vary depending on the type ofspecific binding protein and the specific condition being treated butcan be readily determined by the skilled artisan. In general, regulatoryagencies require that a protein reagent to be used as a therapeutic isformulated so as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

Therapeutic compositions comprising the antigen binding proteins of thepresent disclosure may be administered with a pharmaceuticallyacceptable diluent, carrier, or excipient, in unit dosage form.Administration may be parenteral (e.g., intravenous, subcutaneous),oral, or topical, as non-limiting examples. In addition, any genetherapy technique, using nucleic acids encoding the polypeptides of theinvention, may be employed, such as naked DNA delivery, recombinantgenes and vectors, cell-based delivery, including ex vivo manipulationof patients' cells, and the like.

Thus, an anti-OX40 antibody, or antigen binding portion thereof, of theinvention can be incorporated into a pharmaceutical composition suitablefor parenteral administration. Preferably, the anti-OX40 antibody, orantigen binding portion thereof, will be prepared as an injectablesolution containing 0.1-250 mg/ml antibody. The injectable solution canbe composed of either a liquid or lyophilized dosage form in a flint oramber vial, ampoule or pre-filled syringe. The buffer can be L-histidine(1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Othersuitable buffers include but are not limited to, sodium succinate,sodium citrate, sodium phosphate or potassium phosphate. Sodium chloridecan be used to modify the toxicity of the solution at a concentration of0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectantscan be included for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trehaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRIJ surfactants.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, an anti-OX40 antibody, or antigen binding portion thereof,described herein is administered by intravenous infusion or injection.In another preferred embodiment, an anti-OX40 antibody, or antigenbinding portion thereof, is administered by intramuscular orsubcutaneous injection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The anti-OX40 antibody, or antigen binding portion thereof, of thepresent invention can be administered by a variety of methods known inthe art, although for many therapeutic applications, the preferredroute/mode of administration is subcutaneous injection, intravenousinjection, or infusion. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., (Marcel Dekker, Inc., New York, 1978).

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary.

In certain embodiments, the disclosed antibodies are administered byinhalation, but aerosolization of full IgG antibodies may prove limitingdue to their molecular size (˜150 kDa). To maximize available commercialaerosolization devices, smaller Fab fragments may be required.

In certain embodiments, the subject anti-OX40 antibodies or antibodyfragments of the invention can be used alone.

Diagnostics and Kits

In certain embodiments, any of the anti-OX40 antibodies provided hereinis useful for detecting the presence of OX40 in a biological sample. Incertain embodiments, a biological sample comprises a cell or tissue,such as a sample of a tumor (e.g., NSCLC or breast tumor).

In one embodiment, an anti-OX40 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of OX40 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-OX40 antibody as described herein under conditionspermissive for binding of the anti-OX40 antibody to OX40, and detectingwhether a complex is formed between the anti-OX40 antibody and OX40.Such method may be an in vitro or in vivo method. In one embodiment, ananti-OX40 antibody is used to select subjects eligible for therapy withan anti-OX40 antibody, e.g. where OX40 is a biomarker for selection ofpatients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include cancer.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to OX40. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present disclosurecan be utilized in enzyme immunoassays, wherein the subject polypeptidesare conjugated to an enzyme. When a biological sample comprising an OX40protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the OX40 protein. In oneembodiment, a sample containing cells expressing an OX40 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing an OX40protein recognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of an OX40protein in a biological sample using the antibodies or fragments thereofof the invention can also be prepared. Such kits will include an OX40binding polypeptide, e.g., antibodies or fragments thereof, which bindsto an OX40 protein or portion of said receptor, as well as one or moreancillary reagents suitable for detecting the presence of a complexbetween the binding polypeptide and the receptor protein or portionsthereof. The polypeptide compositions of the present invention can beprovided in lyophilized form, either alone or in combination withadditional antibodies specific for other epitopes. The bindingpolypeptides and/or antibodies, which can be labeled or unlabeled, canbe included in the kits with adjunct ingredients (e.g., buffers, such asTris, phosphate and carbonate, stabilizers, excipients, biocides and/orinert proteins, e.g., bovine serum albumin). For example, the bindingpolypeptides and/or antibodies can be provided as a lyophilized mixturewith the adjunct ingredients, or the adjunct ingredients can beseparately provided for combination by the user. Generally these adjunctmaterials will be present in less than about 5% weight based on theamount of active binding polypeptide or antibody, and usually will bepresent in a total amount of at least about 0.001% weight based onpolypeptide or antibody concentration. Where a second antibody capableof binding to the binding polypeptide is employed, such antibody can beprovided in the kit, for instance in a separate vial or container. Thesecond antibody, if present, is typically labeled, and can be formulatedin an analogous manner with the antibody formulations described above.

Other embodiments are described in the following non-limiting Examples.

Example 1

Experiments were performed to determine the binding capability ofvarious anti-OX40 antibodies. Clones from the anti-OX40 antibody librarywere tested for binding on anti-CD3/anti-CD28 pre-activated humanperipheral blood mononuclear cell (PBMC) for 3 days. Each clone wasincubated at 5 μg/ml with 3×10⁵ pre-activated human PBMCs. The bindingof anti-OX40 antibodies to activated human T cells was revealed by flowcytometry using an APC-labelled anti-human Fc secondary antibody. Aphycoerythrin (PE)-labelled anti-hCD3 antibody was also used to showthat the fluorescence was detected on the T-cells. Secondary antibodyalone was used as a negative control. A commercially availableAPC-labelled anti-human OX40 (clone Ber-ACT35) was used as positivecontrol. The results are shown in FIG. 1. FIG. 1 shows the binding ofanti-OX40 antibody clones on activated human T-cells measured by thepercent CD3+OX40+ T cells. The anti-OX40 clones that were tested areshown on the x-axis. FIG. 1 shows that most of the tested anti-OX40clones are bound on T cells.

Example 2

Experiments were performed to identify OX40 antibody clones with theability to activate the NFkB signaling pathway. OX40⁺HEK293-NFkB-luciferase reporter stable cells were counted andre-suspended in RPMI-1640+5% FCS (complete medium). 6.7×10⁴ cells/well(in 60 μl) were distributed in a white flat-bottom 96-well microplate.The cells were seeded in triplicate. Cells were stimulated by adding 60μl of one of the following reagents: (1) Soluble TNFα (Biolegend; Ref.570102; Lot. B204173) as a positive control; (2) Soluble anti-Histidine(Biolegend; Ref. 652502) at 5 μg/ml+purified rhOX40L (Biolegend; Ref.555704 or R&D systems; Ref. 1054-OX-010) at 1 μg/ml as a positivecontrol; (3) Polyclonal anti-OX40 antibody, used to determine whether amixture of antibodies recognizing various epitopes on the cognateantigen will be better agonists than the monoclonal antibodies; (4)Isotype Control FeH3 antibody at 20 μg/ml; (5) Anti-OX40 antibody clonesat 20 μg/ml. Cells were then incubated at 37° C. in 5% CO₂.

After ˜6 h stimulation the luciferase activity was revealed by theaddition of 100 μl/well of BIO-GLO Luciferase Assay system from Promega(Cat. No. G7941; Lot. 0000168497). The plate was then incubated at roomtemperature in the dark for 5 min under slow shaking conditions.Luciferase activity was obtained by reading with the FlexStation3 fromMolecular Devices (luminescence reading, 500 ms). Results are shown asrelative light units (RLUs).

FIG. 2A shows the luciferase activity measured by RLUs for the controlexperimental conditions after 6 hours of stimulation. As shown in FIG.2A, unstimulated cells, isotype control FeH3 antibody (Ctrl FeH3) andpolyclonal anti-OX40 antibody did not show an increase in RLUs,indicating that the NFkB pathway was not activated. The positivecontrols, soluble anti-Histidine+purified rhOX40L (rhOX40L+αHis) andsoluble TNFα, showed higher RLUs, indicating an increase in NFkBactivity, as expected. FIG. 2B shows luciferase activity measured byRLUs for the anti-OX40 antibody clones tested after 6 hours ofstimulation. Unstimulated cells and isotype control FeH3 antibody (CtrlFeH3) are shown as negative controls. Soluble anti-Histidine+purifiedrhOX40L (rhOX40L+αHis) is shown as a positive control. As shown in FIG.2B, all of the OX40 clones showed an increase in RFUs over the negativecontrol values. These results demonstrate that the OX40/HEK293-NFkB-Lucreporter assay worked properly based on internal controls. Clones 1C4,4D7 1D9 and 1B2 showed the strongest NFkB activation (>50% of signalobserved with purified rhOX40L+anti-His).

A similar set of experiments was carried out, where the stimulation timeof the cells with the various reagents was increased from 6 hours to 15hours. Similar to the first set of experiments, OX40⁺HEK293-NFkB-luciferase reporter stable cells were counted andresuspended in RPMI-1640+5% FCS (complete medium). 5×10⁴ cells/well (in600) were distributed in a white flat-bottom 96-well plate. The cellswere seeded in triplicate. Cells were stimulated by adding 60 μl of oneof the following reagents: (1) Soluble TNFα (biolegend; Ref. 570102;Lot. B204173) as a positive control; (2) Soluble anti-Histidine(Biolegend; Ref. 652502) at 5 μg/ml+purified rhOX40L (Biolegend; Ref.555704) at 1 μg/ml as a positive control; (3) Isotype Control FeH3antibody at 20 μg/ml; (4) Anti-OX40 antibody clones at 20 μg/ml. Cellswere then incubated at 37° C. in 5% CO₂.

After ˜15 h stimulation the luciferase activity was reveal by adding 100μl/well of BIOGLO Luciferase Assay system from Promega (Cat. No. G7941;Lot. 0000168497). The plate was incubated at room temperature in thedark for 5 min under slow shaking conditions. Luciferase activity wasobtained by reading with the FlexStation3 from Molecular Devices(luminescence reading, 500 ms). Results are shown as relative lightunits (RLUs).

FIG. 3A shows the luciferase activity measured by RLUs for the controlexperimental conditions after ˜15 hours of stimulation. As shown in FIG.3A, unstimulated cells, isotype control FeH3 antibody (Ctrl FeH3) andpolyclonal anti-OX40 antibody did not show an increase in RLUs,indicating that the NFkB pathway was not activated. The positivecontrols, soluble anti-Histidine+purified rhOX40L (rhOX40L+αHis) andsoluble TNFα, showed higher RLUs, indicating an increase in NFkBactivity, as expected. FIG. 3B shows luciferase activity measured byRLUs for the anti-OX40 antibody clones tested after ˜15 hours ofstimulation. Unstimulated cells and isotype control FeH3 antibody (CtrlFeH3) are shown as negative controls. Soluble anti-Histidine+purifiedrhOX40L (rhOX40L+αHis) is shown as a positive control. As shown in FIG.3B, all of the OX40 clones showed an increase in RFUs over the negativecontrol values. These results demonstrate that the OX40/HEK293-NFkB-Lucreporter assay worked properly based on internal controls. A prolongedincubation time (15 h instead of 6 h) induced a stronger NFkB activity,suggesting that some clones take longer than 6 hours to stimulate NFkBactivity. In particular clones 1A11, 1C4, 4D7, 1D9, 5C11, 2B4, 4D9, 3C10and 1B2 were among the most active.

Example 3

A human T cell activation assay was used to identify OX40 clones withagonistic activity. A flat-bottom 96-well plate was coated with anti-CD3(clone OKT3) at 1.5 μg/ml+(anti-CD28 or anti-OX40 or anti-CD137 clones)at 10 μg/ml (100 μl/well final) in PBS 1×, overnight at 4° C. Anti-CD3alone and FeD2, an isotype control antibody, were used as negativecontrols. Anti-CD28 was used as a positive control. The next day, theplate was washed three times with 150 μl of sterile complete medium perwell (RPMI1640+10% FCS+Pen/Strep) under a sterile hood. Purified human Tcells were plated at 4×10⁵ cells/well in complete medium. The plate wasincubated at 37° C. for 3 days in a humidified tissue culture incubator.

After 3 days the cells were transferred into a V-bottom 96-well plateand washed twice with cold FACS buffer (PBS1×+2% FCS), and then stainedwith PE-labelled anti-human CD25 (clone M-A251) at 5 μl/well in 70 μl ofFACS buffer for 20 min at 4° C. The cells were spun at 1,500 rpm for 2min and the supernatant was removed by quickly flipping the plate. Next,the cells were washed with 170 μl well of FACS buffer. The cells werespun at 1,500 rpm for 2 min and the supernatant was removed by quicklyflipping the plate. The washing step was repeated twice. The percentageof CD3+CD25+ activated T cells was measured by flow cytometry. As shownin FIG. 4A and FIG. 4B, a number of anti-OX40 clones showed significantT cell co-stimulation. Among those clones showing T cell co-stimulation,clones 2B4, 4D7, 2B3, 4G9 and 1B1 showed the most robust agonistactivity.

Taken together, the results from these experiments have identifiedparticular candidate agonist clones, including 1A11, 2E5, 1B1, 4G9, 5C1,4D7, 5B9, 1C4, 2B4 and 2B 3.

TABLE 1 Sequence Listing Heavy chain variable Light chain variabledomain regions domain regions Ox1A11 EVQLVESGGGLVQTGGSLRLSCAASGDVVMTQSPSSLSASVGDRVTITCRAS FTFSSYWMSWVRQAPGKGLEWVANIKQGIRNDLHWYQQRPGKAPNLLIYAAS QDGSEKYYVDSVKGRFTISRDNAKNSSLHSGVPSRFSGSGSGTDFTLTIDSL LYLQMNSLRAEDTAVYYCARDDYYYGQPEDFATYYCQQANSFPITFGQGTRL MDVWGQGTTVTVSS SEQ ID NO. EIK SEQ ID NO. 2 1Ox1A11 HC CDR1 LC CDR1 SYWMS RASQGIRNDLH SEQ ID NO. 49 SEQ ID NO. 52HC CDR2 LC CDR2 NIKQDGSEKYYVDSVKG AASSLHS SEQ ID NO. 50 SEQ ID NO. 53HC CDR3 LC CDR3 DDYYYGMDV QQANSFPIT SEQ ID NO. 51 SEQ ID NO. 54 Ox1B1QVQLVQSGAEVKKPGASVKVSCKASG QSVLTQPPSASGTPGQRVTISCSGSSYTFTSYYMHWVRQAPGQGLEWMGIIN SNIGSNYVYWYQQLPGTAPKLLIYRNPSGGSTSYAQKFQGRVTMTRDTSTST NQRPSGVPDRFSGSKSGTSASLAISGVYMELSSLRSDDTAVYYCARDPYSSS LRSEDEADYYCAAWDDSLSGLVFGGGWYGAEYFQHWGQGTLVTVSS SEQ TKLTVL SEQ ID NO. 4 ID NO. 3 Ox1B1 HC CDR1LC CDR1 SYYMH SGSSSNIGSNYVY SEQ ID NO. 55 SEQ ID NO. 58 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG RNNQRPS SEQ ID NO. 56 SEQ ID NO. 59 HC CDR3 LC CDR3DPYSSSWYGAEYFQH AAWDDSLSGLV SEQ ID NO. 57 SEQ ID NO. 60 Ox1B2QVQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPSSLSASVGDRVTITCRASYTFTSYYMHWVRQAPGQGLEWMGIIN QSISSYLNWYQQKPGKAPKLLIYAASPSGGSTSYAQKFQGRVTMTRDTSTST SLQSGVPSRFSGSGSGTDFTLTISSLVYMELSSLRSEDTAVYYCARDYYDSS QPEDFAIYYCQQNYNTRQVTFGQGTRGYSDYGMDVWGQGTTVTVSS SEQ LEIK SEQ ID NO. 6 ID NO. 5 Ox1B2 HC CDR1LC CDR1 SYYMH RASQSISSYLN SEQ ID NO. 61 SEQ ID NO. 64 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG AASSLQS SEQ ID NO. 62 SEQ ID NO. 65 HC CDR3 LC CDR3DYYDSSGYSDYGMDV QQNYNTRQVT SEQ ID NO. 63 SEQ ID NO. 66 Ox1B3QVQLVQSGAEVKKPGASVKVSCKTSG AIQLTQSPSSLSASVGDRVTITCRASYTFTGYYLHWVRQAPGQGLEWMGIIN QGISSALAWYQQKPGKAPKVLIYDASPSDGGTRYAQKFQDRVTMTRDMSTST SLESGVPSRFSGSGSGTDFTLTISSLVYMELSSLRPEDTAVYYCARDLEYIG QPEDFATYYCQQFNNYPLTFGGGTKVSGSLSWFDPWGQGTLVTVSS SEQ EIK SEQ ID NO. 8 ID NO. 7 Ox1B3 HC CDR1 LC CDR1GYYLH RASQGISSALA SEQ ID NO. 67 SEQ ID NO. 70 HC CDR2 LC CDR2IINPSDGGTRYAQKFQD DASSLES SEQ ID NO. 68 SEQ ID NO. 71 HC CDR3 LC CDR3DLEYIGSGSLSWFDP QQFNNYPLT SEQ ID NO. 69 SEQ ID NO. 72 Ox1C4EVQLVESGAEVKKPGASVKVSCKTSG LPVLTQPASVSGSPGQSITISCSGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDFLTYDLVSWYKQQPGKAPKLMIYDPGDGSTRNAQKFEGRVTMTRDTSTST VNKRPSGVSDRFSGSKSGNTASLTISVYMELSSLSPEDTAVYYCARDLEYIG GLQAADEADYYCSSYTSSSTPYVFGTSGSLSWFDPWGQGTLVTVSS SEQ GTKVTVL SEQ ID NO. 10 ID NO. 9 Ox1C4 HC CDR1LC CDR1 GYYLH SGTSSDFLTYDLVS SEQ ID NO. 73 SEQ ID NO. 76 HC CDR2 LC CDR2IINPGDGSTRNAQKFEG DVNKRPS SEQ ID NO. 74 SEQ ID NO. 77 HC CDR3 LC CDR3DLEYIGSGSLSWFDP SSYTSSSTPYV SEQ ID NO. 75 SEQ ID NO. 78 Ox1C5QVQLVQSGAEVKKPGASVKVSCKASG QSVLTQPRSVSGSPGQSVTISCTGTSYTFTSYYMHWVRQAPGQGLEWMGIIN SDGGDYNYVSWYQQHPGQAPKLLIYEPSGGSTSYAQKFQGRVTMTTDTSTST VSNRPSGVSNRFSGSKSGNTASLTISAYMELRSLRSDDTAVYYCARDPYSSS GLQAEDEADYYCSSYTSSSTLVVFGGWYGAEYFQHWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 12 ID NO. 11 Ox1C5 HC CDR1LC CDR1 SYYMH TGTSSDGGDYNYVS SEQ ID NO. 79 SEQ ID NO. 82 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG EVSNRPS SEQ ID NO. 80 SEQ ID NO. 83 HC CDR3 LC CDR3DPYSSSWYGAEYFQH SSYTSSSTLVV SEQ ID NO. 81 SEQ ID NO. 84 Ox1D9QVQLVQSGAEVKKPGASVKVSCKASG DVVMTQSPSSLSASVGDRVTITCQASYTFTSYGISWVRQALGQRLEWLGWIN QDISNYLNWYQQKPGKAPKLLIYDASAGDGETKYSPKFQGRVTITRDTSAST NLETGVPSRFSGGGSGTDFTFTISSLAYMDLSGLTSEDTAVYYCARDFLSTM QPEDIATYYCQQFDNLPYTFGQGTKVDYWGQGTLVTVSS SEQ ID NO. EIK SEQ ID NO. 14 13 Ox1D9 HC CDR1 LC CDR1SYGIS QASQDISNYLN SEQ ID NO. 85 SEQ ID NO. 88 HC CDR2 LC CDR2WINAGDGETKYSPKFQG DASNLET SEQ ID NO. 86 SEQ ID NO. 89 HC CDR3 LC CDR3DFLSTMDY QQFDNLPYT SEQ ID NO. 87 SEQ ID NO. 90 Ox1E10EVQLVESGAEVKKPGASVKVSCKTSG QPVLTQPPSASGSPGQSVTISCTGTSYTFTGYYLHWVRQAPGQGLQWMGWIN SDIGGYNYVSWYQQHPGKAPKLLIYEPTSGDTNYAPEYQGRVTMTRDTSIST VSKRPSGVPARFAGSKSGNTASLTVSAYMELSSLRSDDTAVYYCARGHDYSR GLQAEDEADYYCSSYAGNNNHVFGTGTPVGAEALDYWGQGTLVTVSS SEQ TKLTVL SEQ ID NO. 16 ID NO. 15 Ox1E10 HC CDR1LC CDR1 GYYLH TGTSSDIGGYNYVS SEQ ID NO. 91 SEQ ID NO. 94 HC CDR2 LC CDR2WINPTSGDTNYAPEYQG EVSKRPS SEQ ID NO. 92 SEQ ID NO. 95 HC CDR3 LC CDR3GHDYSRTPVGAEALDY SSYAGNNNHV SEQ ID NO. 93 SEQ ID NO. 96 Ox1E7QVQLVQSGAEVKKPGASVKVSCKTSG QSVLTQPASVSGSPGQSITIPCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN NDIGTYNLASWYQHHAGKAPKLIIYDPSDGGTRYAQKFQDRVTMTRDMSTST LNHRPSGVSNRFSGYKSDNTAFLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQPEDESNYYCSSYTMNTTPILFGGSGSLSWFDPWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 17 ID NO. 7 Ox1E7 HC CDR1LC CDR1 GYYLH TGTSNDIGTYNLAS SEQ ID NO. 67 SEQ ID NO. 97 HC CDR2 LC CDR2IINPSDGGTRYAQKFQD DLNHRPS SEQ ID NO. 68 SEQ ID NO. 98 HC CDR3 LC CDR3DLEYIGSGSLSWFDP SSYTMNTTPIL SEQ ID NO. 69 SEQ ID NO. 99 Ox1F2QVQLVQSGAEVKKPGASVKVSCKASG QPVLTQPPSASGTPGQRVTISCSGSSYTFTSYYMHWVRQAPGQGLEWMGIIN SNIGNNFVYWYQQLPGMAPKLLIYKNPSGGSTSYAQKFQGRVTMTRDTSTST NQRPSGVPDRFSGSKSGTSASLAISGVYMELSSLRSDDTAVYYCARDPYSSS LRSEDEADYHCAAWDDSLSGHVVFGGWYGAEYFQHWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 18 ID NO. 3 Ox1F2 HC CDR1LC CDR1 SYYMH SGSSSNIGNNFVY SEQ ID NO. 55 SEQ ID NO. 100 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG KNNQRPS SEQ ID NO. 56 SEQ ID NO. 101 HC CDR3 LC CDR3DPYSSSWYGAEYFQH AAWDDSLSGHVV SEQ ID NO. 57 SEQ ID NO. 102 Ox1G9QVQLVQSGAEVKKPGASVKVSCKTSG QSVLTQPASVSGSPGQSVTVSCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDIGAYESVSWYQQHPGKGPKLIIYDPSDGSTRNAQKFEGRVTMTRDTSTST VSSRPSGVSIRFSGSKSGNSASLTISVYMELSSLSPEDTAVYYCARDLEYIG GLQAEDEAEYFCSSFTRGSTPYVFGTSGSLSWFDPWGQGTLVTVSS SEQ GTKVTVL SEQ ID NO. 20 ID NO. 19 Ox1G9 HC CDR1LC CDR1 GYYLH TGTSSDIGAYESVS SEQ ID NO. 103 SEQ ID NO. 106 HC CDR2LC CDR2 IINPSDGSTRNAQKFEG DVSSRPS SEQ ID NO. 104 SEQ ID NO. 107 HC CDR3LC CDR3 DLEYIGSGSLSWFDP SSFTRGSTPYV SEQ ID NO. 105 SEQ ID NO. 108 Ox2B12QVQLVQSGAEVKKPGASVKVSCKASG QSVLTQPASVSGSPGQSITISCTGTSYTFSNYYMHWVRQAPGQGLEWMGLLN SDVGAYNYVSWYQQHPGRAPKLMIYDPSGGYTTYAQRFQGRVTMTWDTSTST VSDRPSGVSNRFSGSKSGNTASLTISVYMELSSLTSEDTAVYYCAKDPYSSS GLQAEDEADYYCSSYTSSSSLYVFGTWYGAEYFQHWGQGTLVTVSS SEQ GTKVTVL SEQ ID NO. 22 ID NO. 21 Ox2B12 HC CDR1LC CDR1 NYYMH TGTSSDVGAYNYVS SEQ ID NO. 109 SEQ ID NO. 112 HC CDR2LC CDR2 LLNPSGGYTTYAQRFQG DVSDRPS SEQ ID NO. 110 SEQ ID NO. 113 HC CDR3LC CDR3 DPYSSSWYGAEYFQH SSYTSSSSLYV SEQ ID NO. 111 SEQ ID NO. 114 Ox2B3QVQLVQSGAEVKKPGASVKVSCKTSG QSVLTQPASVSGSPGQSITISCTGSSYTFTGYYLHWVRQAPGQGLEWMGIIN SDIGGYNSVSWYQQYPGKAPKLMIHDPSDGGTRYAQKFQDRVTMTRDMSTST VNERPSGISDRFSGSKSGNTASLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQGEDEAEYYCASYSVFSPFLFGRGSGSLSWFDPWGQGTLVTVSS SEQ TKLTVL SEQ ID NO. 23 ID NO. 7 Ox2B3 HC CDR1LC CDR1 GYYLH TGSSSDIGGYNSVS SEQ ID NO. 67 SEQ ID NO. 115 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVNERPS SEQ ID NO. 68 SEQ ID NO. 116 HC CDR3LC CDR3 DLEYIGSGSLSWFDP ASYSVFSPFL SEQ ID NO. 69 SEQ ID NO. 117 Ox2B4QMQLVQSGAEVKKPGASVKVSCKTSG QPVLTQPASVSGSPGQSITISCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDLGAYDYVSWYQQQPGQAPKLIIYDPSDGSTRNAQKFEGRVTMTRDTSTST VNNRPSGVSNRFSGSKSGNTASLTISVYMELSSLSPEDTAVYYCARDLEYIG GLQAEDEADYYCSSYTSSSTLVYVFGSGSLSWFDPWGQGTLVTVSS SEQ TGTKVTVL SEQ ID NO. 25 ID NO. 24 Ox2B4 HC CDR1LC CDR1 GYYLH TGTSSDLGAYDYVS SEQ ID NO. 118 SEQ ID NO. 121 HC CDR2LC CDR2 IINPSDGSTRNAQKFEG DVNNRPS SEQ ID NO. 119 SEQ ID NO. 122 HC CDR3LC CDR3 DLEYIGSGSLSWFDP SSYTSSSTLVYV SEQ ID NO. 120 SEQ ID NO. 123 Ox2B6QVQLVQSGAEVKKPGASVKVSCKTSG LPVLTQPASVSGSPGQSITISCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDVGYYDSVSWYQQYPGKAPKLLIYDPSDGGTRYAQKFQDRVTMTRDMSTST VSKRPSGVSNRFSGSKSGNTASLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQADDEAEYHCSSYSDSSPFVFGTGSGSLSWFDPWGQGTLVTVSS SEQ TKVTVL SEQ ID NO. 26 ID NO. 7 Ox2B6 HC CDR1LC CDR1 GYYLH TGTSSDVGYYDSVS SEQ ID NO. 67 SEQ ID NO. 124 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVSKRPS SEQ ID NO. 68 SEQ ID NO. 125 HC CDR3LC CDR3 DLEYIGSGSLSWFDP SSYSDSSPFV SEQ ID NO. 69 SEQ ID NO. 126 Ox2F2QVQLVQSGAEVKKPGASVKVSCKTSG LPVLTQPASVSGSPGQSITISCTGASYTFTGYYLHWVRQAPGQGLEWMGIIN SDVGGYNSVSWYQQHPGKAPKLMIYDPSDGGTRYAQKFQDRVTMTRDMSTST VSNRPSGISNRFSGSKSGNTASLTVSVYMELSSLRPEDTAVYYCARDLEYIG GLQAEDEADYYCSSYAGSNIVYVFGTSGSLSWFDPWGQGTLVTVSS SEQ GTKVTVL SEQ ID NO. 27 ID NO. 7 Ox2F2 HC CDR1LC CDR1 GYYLH TGASSDVGGYNSVS SEQ ID NO. 67 SEQ ID NO. 127 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVSNRPS SEQ ID NO. 68 SEQ ID NO. 128 HC CDR3LC CDR3 DLEYIGSGSLSWFDP SSYAGSNIVYV SEQ ID NO. 69 SEQ ID NO. 129 Ox2G2QVQLVQSGAEVKKPGASVKVSCKTSG QPVLTQPASVSGSPGQSITISCTGTTYTFTGYYLHWVRQAPGQGLEWMGIIN SDIGGYNSVSWYQQHPGRAPKLIIYDPSDGGTRYAQKFQDRVTMTRDMSTST VTYRPSGVSNRFSGSKSGNTASLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQAEDEADYYCSSYTSGNSVYVFGTSGSLSWFDPWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 28 ID NO. 7 Ox2G2 HC CDR1LC CDR1 GYYLH TGTTSDIGGYNSVS SEQ ID NO. 67 SEQ ID NO. 130 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVTYRPS SEQ ID NO. 68 SEQ ID NO. 131 HC CDR3LC CDR3 DLEYIGSGSLSWFDP SSYTSGNSVYV SEQ ID NO. 69 SEQ ID NO. 132 Ox3C10EVQLVQSGAEVKKPGASVKVSCKASG EIVLTQSPSSLSASVGDRVTITCRASYTFTSYYMHWVRQAPGQGLEWMGIIN QDISSALAWYQQKPGEPPNLLIYDASPSGGSTSYAQKFQGRVTMTRDTSTST TLEGGVPSRFSGSGSGTDFTLTISSLVYMELSSLRSEDTAVYYCARDFSSWY QPEDFATYSCQQFRTYPLTFGGGTKLAYGMDVWGQGTTVTVSS SEQ ID EIK SEQ ID NO. 30 NO. 29 Ox3C10 HC CDR1 LC CDR1SYYMH RASQDISSALA SEQ ID NO. 133 SEQ ID NO. 136 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG DASTLEG SEQ ID NO. 134 SEQ ID NO. 137 HC CDR3 LC CDR3DFSSWYAYGMDV QQFRTYPLT SEQ ID NO. 135 SEQ ID NO. 138 Ox4A11EVQLLESGAEVKKPGASVKVSCKASG QPVLTQPPSVSAAPGQKVTISCSGSSYTFTSYYMHWVRQAPGQGLEWMGIIN SNIGNNYVSWYQQLPGTAPKLLIYDNPSGGSTSYAQKFQGRVTMTRDTSTST DKRPSGIPDRFSGSTSGTSATLGIAGVYMELSSLRSEDTAVYYCARSTLWFS LQTGDEADYYCGTWDSSLGWVFGGGTEFDYWGQGTLVTVSS SEQ ID KLTVL SEQ ID NO. 32 NO. 31 Ox4A11 HC CDR1 LC CDR1SYYMH SGSSSNIGNNYVS SEQ ID NO. 139 SEQ ID NO. 142 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG DNDKRPS SEQ ID NO. 140 SEQ ID NO. 143 HC CDR3 LC CDR3STLWFSEFDY GTWDSSLGWV SEQ ID NO. 141 SEQ ID NO. 144 Ox4A12QVQLVQSGAEVKKPGASVKVSCKASG QSVLTQPRSVSGSPGQSVTISCTGTSYTFTSYYMHWVRQAPGQGLEWMGIIN SDGGDYNYVSWYQQHPGQAPKLLIYEPSGGSTSYAQKFQGRVTMTRDTSTST VSNRPSGVSNRFSGSKSGNTASLTISVYMELSSLRSDDTAVYYCARDPYSSS GLQAEDEADYYCSSYTSSSTLVVFGGWYGAEYFQHWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 33 ID NO. 3 Ox4A12 HC CDR1LC CDR1 SYYMH TGTSSDGGDYNYVS SEQ ID NO. 55 SEQ ID NO. 145 HC CDR2LC CDR2 IINPSGGSTSYAQKFQG EVSNRPS SEQ ID NO. 56 SEQ ID NO. 146 HC CDR3LC CDR3 DPYSSSWYGAEYFQH SSYTSSSTLVV SEQ ID NO. 57 SEQ ID NO. 147 Ox4B6EVQLVQSGAEVKKPGASVKVSCKASG EIVLTQSPSSLSASVGDRVTITCRASYTFTSYYMHWVRQAPGQGLEWMGIIN QDISSALAWYQQKPGEPPNLLIYDASPSGGSTSYAQKFQGRVTMTRDTSTST TLEGGVPSRFSGSGSGTDFTLTISSLVYMELSSLRSEDTAVYYCARDFSSWY QPEDFATYSCQQFRTYPLTFGGGTKLAYGMDVWGQGTTVTVSS SEQ ID EIK SEQ ID NO. 35 NO. 34 Ox4B6 HC CDR1 LC CDR1SYYMH RASQDISSALA SEQ ID NO. 148 SEQ ID NO. 151 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG DASTLEG SEQ ID NO. 149 SEQ ID NO. 152 HC CDR3 LC CDR3DFSSWYAYGMDV QQFRTYPLT SEQ ID NO. 150 SEQ ID NO. 153 Ox4D4QVQLVQSGAEVKKPGASVKVSCKTSG LPVLTQPASVSGSPGQSITISCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDVGAYNSVSWYQQRPGKAPKLMIYDPSDGGTRYAQKFQDRVTMTRDMSTST VIQRPSEVSHRFSGSKSGNTASLTISVYMELSSLRPEDTAVYYCARDLEYIG GLLPEDEAEYFCGSYAASTTFVFGGGSGSLSWFDPWGQGTLVTVSS SEQ TKLTVL SEQ ID NO. 36 ID NO. 7 Ox4D4 HC CDR1LC CDR1 GYYLH TGTSSDVGAYNSVS SEQ ID NO. 67 SEQ ID NO. 154 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVIQRPS SEQ ID NO. 68 SEQ ID NO. 155 HC CDR3LC CDR3 DLEYIGSGSLSWFDP GSYAASTTFV SEQ ID NO. 69 SEQ ID NO. 156 Ox4D7QVQLVQSGAEVKKPGASVKVSCKASG QSVLTQPRSVSGSPGQSVTISCTGTSYTFTSYYMHWVRQAPGQGLEWMGIIN SDGGDYNYVSWYQQHPGQAPKLLIYEPSGGSTSYAQKFQGRVTMTTDTSTST VSNRPSGVSNRFSGSKSGNTASLTISAYMELRSLRSDDTAVYYCARDPYSSS GLQAEDEADYYCSSYTSSSTLVVFGGWYGAEYFQHWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 38 ID NO. 37 Ox4D7 HC CDR1LC CDR1 SYYMH TGTSSDGGDYNYVS SEQ ID NO. 157 SEQ ID NO. 160 HC CDR2LC CDR2 IINPSGGSTSYAQKFQG EVSNRPS SEQ ID NO. 158 SEQ ID NO. 161 HC CDR3LC CDR3 DPYSSSWYGAEYFQH SSYTSSSTLVV SEQ ID NO. 159 SEQ ID NO. 162 Ox4D9EVQLVQSGAEVKKPGASVKLSCKASG QPVLTQPPSASGTPGQRVSISCSGSSYTFTSYFMHWVRQAPGQGLEWMGIIN SNIGTNTVNWYQQLPGTAPKLLVYSNPSGGSTSYAQKFQGRLTMTRDTSTST NQRPSGVPDRFSGSKSGTSASLAISGAYMELRSLRSDDTAVYYCARDPYSSS LQSEDEADYFCSAWDDSLNGQVFGAGWYGAEYFQHWGQGTLVTVSS SEQ TKVTVL SEQ ID NO. 40 ID NO. 39 Ox4D9 HC CDR1LC CDR1 YFMH SGSSSNIGTNTVN SEQ ID NO. 163 SEQ ID NO. 166 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG SNNQRPS SEQ ID NO. 164 SEQ ID NO. 167 HC CDR3 LC CDR3DPYSSSWYGAEYFQH SAWDDSLNGQV SEQ ID NO. 165 SEQ ID NO. 168 Ox4G9QVQLVQSGAEVKKPGASVKVSCKTSG LPVLTQPASVSGSPGQSITISCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDVGYYDSVSWYQQYPGKAPKLLIYDPSDGGTRYAQKFQDRVTMTRDMSTST VSKRPSGVSNRFSGSKSGNTASLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQADDEAEYHCSSYSDSSPFVFGTGSGSLSWFDPWGQGTLVTVSS SEQ TKVTVL SEQ ID NO. 41 ID NO. 7 Ox4G9 HC CDR1LC CDR1 GYYLH TGTSSDVGYYDSVS SEQ ID NO. 67 SEQ ID NO. 169 CDR2 LC CDR2IINPSDGGTRYAQKFQD DVSKRPS SEQ ID NO. 68 SEQ ID NO. 170 HC CDR3 LC CDR3DLEYIGSGSLSWFDP SSYSDSSPFV SEQ ID NO. 69 SEQ ID NO. 171 Ox4H4EVQLVQSGGGLVQPGGSLRLSCAASG DIVMTQSPSSLSASVGDRVTITCQASFSFSGYDMSWVRQAPGKGLEWVSSIS QDISNSLNWYQQKPGKAPNLLIYDASTSGGSTNYADSVNGRFIISRDNSKNT TLQRGVPSRFSGSGSGTKFTFTISSLLYLQMNSLRTEDTAVYYCAREGSGWY QPEDIATYYCQQYANLPPITFGQGTRDAGYFDYWGQGTLVTVSS SEQ ID LEIK SEQ ID NO. 43 NO. 42 Ox4H4 HC CDR1LC CDR1 GYDMS QASQDISNSLN SEQ ID NO. 172 SEQ ID NO. 175 HC CDR2 LC CDR2SISTSGGSTNYADSVNG DASTLQR SEQ ID NO. 173 SEQ ID NO. 176 HC CDR3 LC CDR3EGSGWYDAGYFDY QQYANLPPIT SEQ ID NO. 174 SEQ ID NO. 177 Ox5B9QVQLVQSGAEVKKPGASVKVSCKTSG LPVLTQPASVSGSPGQSITISCTGTTYTFTGYYLHWVRQAPGQGLEWMGIIN SDIGGYNSVSWYQQHPGRAPKLIIYDPSDGGTRYAQKFQDRVTMTRDMSTST VTYRPSGVSNRFSGSKSGNTASLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQAEDEADYYCSSYTSGNSVYVFGTSGSLSWFDPWGQGTLVTVSS SEQ GTKLTVL SEQ ID NO. 45 ID NO. 44 Ox5B9 HC CDR1LC CDR1 GYYLH TGTTSDIGGYNSVS SEQ ID NO. 178 SEQ ID NO. 181 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVTYRPS SEQ ID NO. 179 SEQ ID NO. 182 HC CDR3LC CDR3 DLEYIGSGSLSWFDP SSYTSGNSVYV SEQ ID NO. 180 SEQ ID NO. 183 Ox5C1EVQLVQSGGGLVQPGQSLRLSCTPYG LPVLTQSPSVSVSPGQTGYMNCYGHEFSFNDYGMSWVRQAPGKGLEWLAFVG LTDKYVSWYQKKPGQSPVLVIYEDTKSKASGGASENVAAVQGRFSFSRDDAK RPSGIPDRFSGSNSGDTATLTISGTQGIAYLQLNNLKPEDTGVYFCTRDLGT ALDEADYYCQAWDSNTVIFGGGTKLTSGPYFFDYWGQGTLVTVSS SEQ VL SEQ ID NO. 47 ID NO. 46 Ox5C1 HC CDR1 LC CDR1DYGMS YGHELTDKYVS SEQ ID NO. 184 SEQ ID NO. 187 HC CDR2 LC CDR2FVGSKASGGASENVAAVQG EDTKRPS SEQ ID NO. 185 SEQ ID NO. 188 HC CDR3LC CDR3 DLGTSGPYFFDY QAWDSNTVI SEQ ID NO. 186 SEQ ID NO. 189 Ox5D7QVQLVQSGAEVKKPGASVKVSCKTSG LPVLTQPPSVSAAPGQKVTISCSGSSYTFTGYYLHWVRQAPGQGLEWMGIIN SNIGNNYVSWYQQLPGTAPKLLIYDNPSDGGTRYAQKFQDRVTMTRDMSTST NERPSGIPDRFSGSKSGNTASLTISGVYMELSSLRPEDTAVYYCARDLEYIG LQAEDEADYYCSSYTDRDTPYVFGGGSGSLSWFDPWGQGTLVTVSS SEQ TKVTVL SEQ ID NO. 48 ID NO. 7 Ox5D7 HC CDR1LC CDR1 GYYLH SGSSSNIGNNYVS SEQ ID NO. 67 SEQ ID NO. 190 HC CDR2 LC CDR2IINPSDGGTRYAQKFQD DNNERPS SEQ ID NO. 68 SEQ ID NO. 191 HC CDR3 LC CDR3DLEYIGSGSLSWFDP SSYTDRDTPYV SEQ ID NO. 69 SEQ ID NO. 192 Ox4B5QMQLVQSGAEVKKPGASVKVSCKASG DIVMTQSPSSLSASVGDRVTITCQASYTFTNFFMHWVRQAPGQGLEWMGIIN QDISKYLNWYQQKPGKAPKLLIYDASPSGGSTSYAQKFQGRVTMTRDTSTST NLETGVPSRFSGSGSGTDFSFTISNLVYMELSSLRSEDTAVYYCARDRELLW QPEDIATYYCQQSANLPITFGQGTKVFGELSGAFDIWGQGTMVTVSS EIK SEQ ID NO. 194 SEQ ID NO. 193 Ox4B5 HC CDR1LC CDR1 NFFMH QASQDISKYLN SEQ ID NO. 195 SEQ ID NO. 198 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG DASNLET SEQ ID NO. 196 SEQ ID NO. 199 HC CDR3 LC CDR3DRELLWFGELSGAFDI QQSANLPIT SEQ ID NO. 197 SEQ ID NO. 200 Ox2E5EVQLVQSGAEVKKPGASVTLSCKASG QSVLTQPASVSASPGQSITISCTGASHTFTNYYMHWVRQAPGQGLEWMGIIN SDIGGYDYVSWYQQHSGKAPKLMIYEPGGGGTSYAQKFHDRVAMTRDTSTST ASKRPSGVSHRFSGSKSGNTASLIISVYMELSSLRSEDTAVYYCTRGLYSAY GLQAEDEADYYCSSHTTSSTWVFGGGDSPSDLWGQGTLVTVSS SEQ ID TKLTVL SEQ ID NO. 202 NO. 201 Ox2E5 HC CDR1LC CDR1 NYYMH TGASSDIGGYDYVS SEQ ID NO. 203 SEQ ID NO. 206 HC CDR2LC CDR2 IINPGGGGTSYAQKFHD EASKRPS SEQ ID NO. 204 SEQ ID NO. 207 HC CDR3LC CDR3 GLYSAYDSPSDL SSHTTSSTWV SEQ ID NO. 205 SEQ ID NO. 208 Ox2B5QVQLVQSGAEVKKPGASVKVSCKASG DIQMTQSPSSLSASLGDRVTITCRANYTFTSYYMHWVRQAPGQGLEWMGIIN QSISRYLNWYQHKPGKAPKLLIYAASPSGGSTSYAQKFQGRVTMTRDTSTST SLQSGVPSRFSGNGSGTDFTLTISSLVYMELSSLRSEDTAVYYCARDYYDSS QPEDFATYYCQQSYSTPSITFGQGTRGYSDYGMDVWGQGTTVTVSS SEQ LEIK SEQ ID NO. 209 ID NO. 5 Ox2B5 HC CDR1LC CDR1 SYYMH RANQSISRYLN SEQ ID NO. 61 SEQ ID NO. 210 HC CDR2 LC CDR2IINPSGGSTSYAQKFQG AASSLQS SEQ ID NO. 62 SEQ ID NO. 211 HC CDR3 LC CDR3DYYDSSGYSDYGMDV QQSYSTPSIT SEQ ID NO. 63 SEQ ID NO. 212 Ox5C11QVQLVQSGAEVKKPGASVKVSCKTSG NFMLTQPRSVSGSPGQSVTISCTGTSYTFTGYYLHWVRQAPGQGLEWMGIIN SDIGGYSSVSWYQQHPGKAPKLIIYDPSDGGTRYAQKFQDRVTMTRDMSTST VTERPSGVPDRFSGSKSGDTATLTISVYMELSSLRPEDTAVYYCARDLEYIG GLQAEDEADYFCSSYAGVYTYVFGTGSGSLSWFDPWGQGTLVTVSS SEQ TKVTVL SEQ ID NO. 213 ID NO. 7 Ox5C11 HC CDR1LC CDR1 GYYLH TGTSSDIGGYSSVS SEQ ID NO. 67 SEQ ID NO. 214 HC CDR2LC CDR2 IINPSDGGTRYAQKFQD DVTERPS SEQ ID NO. 68 SEQ ID NO. 215 HC CDR3LC CDR3 DLYIGSGSLSWFDP SSYAGVYTYV SEQ ID NO. 69 SEQ ID NO. 216

I claim:
 1. An isolated anti-OX40 antibody, or an antigen-bindingfragment thereof, wherein the heavy chain variable domain comprises theamino acid sequence of SEQ ID NO: 24; and comprises a light chainvariable domain comprising the amino acid sequence SEQ ID NO:
 25. 2. Theanti-OX40 antibody, or antigen-binding fragment thereof, of claim 1,wherein the antibody is a human antibody.
 3. The anti-OX40 antibody ofclaim 1, which is an IgG.
 4. The anti-OX40 antibody of claim 1, whereinthe antibody is an IgG1, IgG2, IgG3 or an IgG4 isotype.
 5. The anti-OX40antibody, or antigen-binding fragment thereof, of claim 1, wherein theantigen-binding fragment is a Fab fragment or an scFv.
 6. Apharmaceutical composition comprising the anti-OX40 antibody, orantibody fragment of claim 1, and a pharmaceutically acceptable carrier.7. An isolated anti-OX40 antibody, comprising: a heavy chain and a lightchain, wherein (i) the heavy chain comprises: a heavy chaincomplementarity determining region 1 (CDR1) having the amino acidsequence of SEQ ID NO:118, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO:119, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO:120; and (ii) the light chain comprises: a lightchain CDR1 having the amino acid sequence of SEQ ID NO:121, a lightchain CDR2 having the amino acid sequence of SEQ ID NO:122, and a lightchain CDR3 having the amino acid sequence of SEQ ID NO:123.
 8. Theisolated anti-OX40 antibody of claim 7, wherein the antibody is a humanantibody.